Biomarkers for Homologous Recombination Deficiency in Cancer

Biomarkers for Homologous Recombination Deficiency in Cancer Abstract Defective DNA repair is a common hallmark of cancer. Homologous recombination is a DNA repair pathway of clinical interest due to the sensitivity of homologous recombination-deficient cells to poly-ADP ribose polymerase (PARP) inhibitors. The measurement of homologous recombination deficiency (HRD) in cancer is therefore vital to the appropriate design of clinical trials incorporating PARP inhibitors. However, methods to identify HRD in tumors are varied and controversial. Understanding existing and new methods to measure HRD is important to their appropriate use in clinical trials and practice. The aim of this review is to summarize the biology and clinical validation of current methods to measure HRD, to aid decision-making for patient stratification and translational research in PARP inhibitor trials. We discuss the current clinical development of PARP inhibitors, along with established indicators for HRD such as germline BRCA1/2 mutation status and clinical response to platinum-based therapy. We then examine newer assays undergoing clinical validation, including 1) somatic mutations in homologous recombination genes, 2) “genomic scar” assays using array-based comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) analysis or mutational signatures derived from next-generation sequencing, 3) transcriptional profiles of HRD, and 4) phenotypic or functional assays of protein expression and localization. We highlight the strengths and weaknesses of each of these assays, for consideration during the design of studies involving PARP inhibitors. Each cell is equipped with a versatile set of DNA damage response (DDR) networks that guard the genome against mutational insults (1). Double-strand breaks (DSBs) are a particularly hazardous form of DNA damage and are repaired by two major repair pathways—error-free homologous recombination and nonhomologous end-joining (NHEJ) (2,3). Homologous recombination deficiency (HRD) was initially described in cancers with germline mutations of the tumor suppressors BRCA1 and BRCA2 (4). However, genetic and epigenetic inactivation of other homologous recombination components can lead to HRD in sporadic cancers, broadly termed BRCAness (5–7). As homologous recombination is required for the repair of DSBs generated during DNA interstrand cross-link (ICL) resolution (8), homologous recombination-deficient tumors are sensitive to ICL-generating platinum chemotherapy (9). Furthermore, inhibitors of the DNA repair enzyme poly-ADP ribose polymerase 1 (PARP1) (10) have rapidly gained clinical interest following observations that cells with mutant BRCA1 and BRCA2 display exquisite sensitivity to these agents (11,12). This sensitivity of BRCA1/2 mutant cells to PARP inhibition results from the synthetic lethality of cells with defective homologous recombination-mediated DNA repair to toxic replication intermediates generated on chromatin by PARP “trapping” (13). Three PARP inhibitors (olaparib, rucaparib, and niraparib) are currently approved by the Food and Drug Administration (FDA) (Table 1). Biomarkers that predict HRD in BRCA wild-type tumors could theoretically expand the use of PARP inhibitors beyond BRCA mutant tumors. Here we aim to summarize these markers in the context of their biology, clinical validation, and potential clinical utility in trials targeting HRD. Approved HRD Biomarkers for PARP Inhibitor Use Germline BRCA Mutations The clinical evidence so far suggests that germline BRCA (gBRCA) mutations remain the best clinical biomarkers for response to PARP inhibitor therapy (17,21,22), and BRCA mutant cells show clear evidence of HRD in vitro (23,24). PARP inhibitor trials have used distinct sequencing assays to evaluate the presence of gBRCA mutations (25–28). The Myriad Genetics BRACAnalysis CDx platform (Myriad Genetics; Salt Lake City, UT) has been FDA approved to identify ovarian cancer patients with suspected deleterious gBRCA variants eligible for treatment with olaparib. This blood-based assay was validated retrospectively and matched to local results via a bridging study (29). Larger phase III studies of PARP inhibitors in ovarian cancer (study 19 [15] and the NOVA trial [17]) and breast cancer (OlympiAD [21]) subsequently used BRACAnalysis to establish gBRCA mutation status. There are currently no approved diagnostic assays for HRD based on germline mutations of other homologous recombination genes. Using germline mutations in homologous recombination genes to classify tumors as HRD, however, suffers from several drawbacks. Variants of uncertain significance (VUS) remain a problem, as they do for germline testing of tumor suppressor genes toward familial risk prediction, as the genotype-phenotype correlations of many variants are unclear. Somatic reversion mutations may render a tumor functionally homologous recombination proficient even with germline mutations in homologous recombination repair–related genes (30). Ideally, biomarkers of HRD should not only predict HRD in BRCA wild-type tumors, but also refine the BRCA mutant population to account for phenotypic variants and reversions. The absence of BRCA locus–specific LOH, for instance, is associated with poorer platinum response in gBRCA mutant tumors (31). Because of these caveats, there have been efforts to generate surrogate biomarkers of homologous recombination for patient stratification in PARP inhibitor trials. Platinum Sensitivity as a Surrogate Biomarker for HRD Platinum sensitivity in vitro is a feature of homologous recombination-deficient cells, and BRCA mutant ovarian and breast tumors demonstrate increased platinum sensitivity (9,32). As platinum is a key component of firstline chemotherapy in ovarian cancer, prior platinum sensitivity has been evaluated as a surrogate clinical index for prediction of efficacy to PARP inhibition (26). In the phase III NOVA study (17) of niraparib in platinum-sensitive ovarian cancer, the highest benefit was seen in gBRCA mutated patients. However, there was a benefit noted in all subsets of platinum-sensitive ovarian cancer (17), leading to niraparib’s approval for all platinum-sensitive metastatic ovarian cancers. PARP inhibitor sensitivity does not show complete overlap with platinum sensitivity (22). For example, cancers with defects in nucleotide excision repair (NER) may respond to platinum therapy, though this does not confer a concurrent PARP inhibitor sensitivity (33). Conversely, there is also a proportion of platinum-resistant patients who remain sensitive to PARP inhibitors, which would be missed by a “platinum-sensitive only” indication (Figure 1A) (14). For these reasons, it remains pertinent to explore assays that may accurately define HRD status in tumors. Novel Biomarkers of HRD A plethora of markers have been described to confer PARP inhibitor sensitivity in vitro (34,35), but most of these have not been validated clinically in well-annotated data sets. Here we categorize the various methods utilized for HRD diagnosis to date and review their biological and clinical rationale (Figure 1B). Figure 1. View largeDownload slide Homologous recombination deficiency (HRD) in cancers and assays for its estimation. A) The relationship between HRD, platinum, and poly-ADP ribose polymerase (PARP) inhibitor sensitivity in cancer. Most HRD tumors are sensitive to both platinum and PARP inhibition, but these sets do not overlap completely. Most PARP inhibitor–sensitive tumors will be HRD, but some HRD tumors will be PARP inhibitor resistant. HRD tumors may be platinum resistant due to drug efflux pumps and yet retain PARP inhibitor sensitivity. Conversely, tumors may also be platinum sensitive due to defects in nucleotide excision repair, which does not confer synthetic lethality to PARP inhibitors. B) An overview of HRD assays and their biological rationale. Key proteins involved in homologous recombination are depicted in the upper left section, and the color code depicts the nature of their typical alteration in cancer (red: somatic mutations; blue: germline mutations; green: altered gene expression; for clarity, only the most prevalent alteration is color-coded). These alterations lead to functional homologous recombination deficiency, which is reported by downstream assays of homologous recombination. The impact of HRD is the accumulation of specific patterns of mutations across the genome, which are studied using genomic scar assays. DSB = double-strand DNA breaks; HR = homologous recombination; LOH = loss of heterozygosity; LST = large-scale transition; TAI = telomeric allelic imbalance. Figure 1. View largeDownload slide Homologous recombination deficiency (HRD) in cancers and assays for its estimation. A) The relationship between HRD, platinum, and poly-ADP ribose polymerase (PARP) inhibitor sensitivity in cancer. Most HRD tumors are sensitive to both platinum and PARP inhibition, but these sets do not overlap completely. Most PARP inhibitor–sensitive tumors will be HRD, but some HRD tumors will be PARP inhibitor resistant. HRD tumors may be platinum resistant due to drug efflux pumps and yet retain PARP inhibitor sensitivity. Conversely, tumors may also be platinum sensitive due to defects in nucleotide excision repair, which does not confer synthetic lethality to PARP inhibitors. B) An overview of HRD assays and their biological rationale. Key proteins involved in homologous recombination are depicted in the upper left section, and the color code depicts the nature of their typical alteration in cancer (red: somatic mutations; blue: germline mutations; green: altered gene expression; for clarity, only the most prevalent alteration is color-coded). These alterations lead to functional homologous recombination deficiency, which is reported by downstream assays of homologous recombination. The impact of HRD is the accumulation of specific patterns of mutations across the genome, which are studied using genomic scar assays. DSB = double-strand DNA breaks; HR = homologous recombination; LOH = loss of heterozygosity; LST = large-scale transition; TAI = telomeric allelic imbalance. Somatic Mutations in Homologous Recombination Genes Somatic mutations in BRCA genes are more common than gBRCA mutations. In high-grade serous ovarian cancer, approximately 7% of patients have BRCA somatic mutations and approximately 14% have germline mutations (36,37). The tissue-based FoundationFocus CDxBRCA assay (Foundation medicine; Cambridge, MA) examines both germline and somatic mutations in the tumor and is FDA approved as a companion diagnostic to rucaparib based on the ARIEL trials (19,20). In vitro studies show that determinants of PARP inhibitor sensitivity beyond BRCA1 and BRCA2 include proteins involved in homologous recombination and related pathways (38). In vitro criteria that predict sensitivity to PARP inhibitors include 1) defects in recombination substrate assays (39); 2) inability for RAD51 to form distinct nuclear foci required for functional homologous recombination upon exposure to DNA damage (40); and 3) in vitro sensitivity to platinum salts and PARP inhibitors (11). Cancer-associated mutations in genes fulfilling the above criteria, including PALB2, BARD1, BRIP1, RAD51B, RAD51C, RAD51D, ATM, FAAP20, CHEK2, FAN1, FANCE, FANCM, and POLQ (36,41), are potential biomarkers of HRD in cancer but suffer from the same uncertainty over functional significance as germline mutations. Nonetheless, several of these mutations correlate with clinical responses to PARP inhibitor treatment (Table 2), with the TOPARP study (42) in prostate cancer being a notable example. Apart from mutational inactivation, BRCA1 can also be suppressed through promoter methylation. Notably however, BRCA1-methylated ovarian cancer patients do not appear to have a survival benefit with platinum-based chemotherapy (36,43–45) or demonstrate long-term response to PARP inhibitor therapy (46). Table 2. Clinical evidence of PARP inhibitor sensitivity to somatic mutations in homologous recombination genes* Study Design Cancer type Drug Platform to detect mutations Homologous recombination genes studied Results TOPARP (Mateo et al., 2015) (42) Single-arm phase II Prostate Olaparib GeneRead DNAseq Panel (Qiagen, Germany) BRCA1, BRCA2, ATM, FANCA, CHEK2, PALB2, NBN, HDAC2 16 of 50 patients (33%) had tumor aberrations in DNA-repair genes, with 14 of 16 biomarker-positive patients (88%) having a response to olaparib. ARIEL2 (Swisher et al., 2017) (19) Single-arm phase II Ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) BRCA1, BRCA2, ATM, BRIP1, CHEK2, FANCA, FANCI, FANCM, NBN, RAD51C, RAD51D, RAD54L 74% of patients with somatic BRCA mutations had an objective response. Response was also noted in patients with mutations in non-BRCA homologous recombination genes (ATM, NBN, RAD51C, and RAD51D). ARIEL3 (Coleman et al., 2017) (20) Phase III High-grade serous ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) (somatic) BRCA BRCA mutated patients had longer PFS compared with the placebo group (13.6 vs 5.4 months, HR = 0.32, 95% CI = 0.24 to 0.42). BRCAnalysis CDx test (Myriad Genetics, Salt Lake City, UT) (germline) (Multiple other genes on NGS) Study Design Cancer type Drug Platform to detect mutations Homologous recombination genes studied Results TOPARP (Mateo et al., 2015) (42) Single-arm phase II Prostate Olaparib GeneRead DNAseq Panel (Qiagen, Germany) BRCA1, BRCA2, ATM, FANCA, CHEK2, PALB2, NBN, HDAC2 16 of 50 patients (33%) had tumor aberrations in DNA-repair genes, with 14 of 16 biomarker-positive patients (88%) having a response to olaparib. ARIEL2 (Swisher et al., 2017) (19) Single-arm phase II Ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) BRCA1, BRCA2, ATM, BRIP1, CHEK2, FANCA, FANCI, FANCM, NBN, RAD51C, RAD51D, RAD54L 74% of patients with somatic BRCA mutations had an objective response. Response was also noted in patients with mutations in non-BRCA homologous recombination genes (ATM, NBN, RAD51C, and RAD51D). ARIEL3 (Coleman et al., 2017) (20) Phase III High-grade serous ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) (somatic) BRCA BRCA mutated patients had longer PFS compared with the placebo group (13.6 vs 5.4 months, HR = 0.32, 95% CI = 0.24 to 0.42). BRCAnalysis CDx test (Myriad Genetics, Salt Lake City, UT) (germline) (Multiple other genes on NGS) * CI = confidence interval; HR = hazard ratio; NGS = next-generation sequencing; PARP = poly-ADP ribose polymerase; PFS = progression-free survival. Table 2. Clinical evidence of PARP inhibitor sensitivity to somatic mutations in homologous recombination genes* Study Design Cancer type Drug Platform to detect mutations Homologous recombination genes studied Results TOPARP (Mateo et al., 2015) (42) Single-arm phase II Prostate Olaparib GeneRead DNAseq Panel (Qiagen, Germany) BRCA1, BRCA2, ATM, FANCA, CHEK2, PALB2, NBN, HDAC2 16 of 50 patients (33%) had tumor aberrations in DNA-repair genes, with 14 of 16 biomarker-positive patients (88%) having a response to olaparib. ARIEL2 (Swisher et al., 2017) (19) Single-arm phase II Ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) BRCA1, BRCA2, ATM, BRIP1, CHEK2, FANCA, FANCI, FANCM, NBN, RAD51C, RAD51D, RAD54L 74% of patients with somatic BRCA mutations had an objective response. Response was also noted in patients with mutations in non-BRCA homologous recombination genes (ATM, NBN, RAD51C, and RAD51D). ARIEL3 (Coleman et al., 2017) (20) Phase III High-grade serous ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) (somatic) BRCA BRCA mutated patients had longer PFS compared with the placebo group (13.6 vs 5.4 months, HR = 0.32, 95% CI = 0.24 to 0.42). BRCAnalysis CDx test (Myriad Genetics, Salt Lake City, UT) (germline) (Multiple other genes on NGS) Study Design Cancer type Drug Platform to detect mutations Homologous recombination genes studied Results TOPARP (Mateo et al., 2015) (42) Single-arm phase II Prostate Olaparib GeneRead DNAseq Panel (Qiagen, Germany) BRCA1, BRCA2, ATM, FANCA, CHEK2, PALB2, NBN, HDAC2 16 of 50 patients (33%) had tumor aberrations in DNA-repair genes, with 14 of 16 biomarker-positive patients (88%) having a response to olaparib. ARIEL2 (Swisher et al., 2017) (19) Single-arm phase II Ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) BRCA1, BRCA2, ATM, BRIP1, CHEK2, FANCA, FANCI, FANCM, NBN, RAD51C, RAD51D, RAD54L 74% of patients with somatic BRCA mutations had an objective response. Response was also noted in patients with mutations in non-BRCA homologous recombination genes (ATM, NBN, RAD51C, and RAD51D). ARIEL3 (Coleman et al., 2017) (20) Phase III High-grade serous ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) (somatic) BRCA BRCA mutated patients had longer PFS compared with the placebo group (13.6 vs 5.4 months, HR = 0.32, 95% CI = 0.24 to 0.42). BRCAnalysis CDx test (Myriad Genetics, Salt Lake City, UT) (germline) (Multiple other genes on NGS) * CI = confidence interval; HR = hazard ratio; NGS = next-generation sequencing; PARP = poly-ADP ribose polymerase; PFS = progression-free survival. The challenge of interpreting somatic alterations in homologous recombination-related genes is highlighted by phosphatase and tensin homolog (PTEN) and HRD (47). PTEN loss of function mutations confer PARP inhibitor sensitivity in endometrial carcinomas and glioblastomas (48,49), but not in prostate cancer cells (50) and ovarian cancer patients (51). This discrepancy highlights the heterogeneity underlying genome maintenance mechanisms, necessitating context-specific clinical evaluation and validation of specific biomarkers. Furthermore, the low frequency and indeterminate functional relevance of specific mutations in non-BRCA homologous recombination genes add further to the difficulties of a single-gene approach. Consequently, efforts have been made to identify genomic, transcriptomic, proteomic, and functional assays that would be reflective of underlying HRD in tumors. Genomic Scar Assays Cancer genomes often harbor chromosomal aberrations arising from defective DNA repair (52). In BRCA mutant cells, chromosomal spreads reveal increased gross chromosomal rearrangements (23). This leads to the development of assays to evaluate the “genomic scar” left behind by the loss of homologous recombination function, irrespective of which component of the pathway was lost. Current genomic scar assays use a combination of high-throughput genomic profiling techniques including array-based comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) genotyping, and next-generation sequencing (NGS). aCGH of Structural Chromosomal Rearrangements aCGH is a technique that detects genomic copy number variations in tumors (53). Using aCGH genomic profiles, primary breast cancers reveal four distinct subgroups, two of which were enriched for BRCA1/2 deficiency (54). The presence of a BRCA1-like aCGH signature predicted favorable response to platinum vs conventional anthracycline-based therapy (55). Only two-thirds of BRCA1-like tumors harbor either the BRCA1 mutation or promoter methylation, suggesting that the signature captures a wider spectrum of HRD tumors. The BRCA1-like and BRCA2-like profiles were later combined to establish a BRCA-likeCGH score (56) and an assay compatible with formalin-fixed paraffin-embedded (FFPE) samples (57). BRCA-likeCGH was evaluated retrospectively in a breast cancer clinical trial comparing chemotherapy alone with chemotherapy followed by high-dose platinum and autologous stem cell transplant. The study showed no difference in survival for the unselected population, whereas BRCA-likeCGH patients had a statistically significant overall survival (OS) benefit from high-dose platinum-based therapy (hazard ratio [HR] = 0.19, 95% confidence interval [CI] = 0.08 to 0.48). aCGH assays have not been evaluated in the context of PARP inhibition. SNP-Based “Genomic Scar” Assays In 2012, three SNP-based assays were developed to quantify the extent of chromosomal abnormalities—telomeric allelic imbalance (TAI) (58), loss of heterozygosity (LOH) (59), and large-scale transition (LST) (60), jointly termed “genomic scar” assays (Table 3). Each genomic scar assay measures a discrete type of gross genomic aberration, and they were developed using a training cohort of tumors from BRCA1/2 mutated patients. In an in silico analysis of 5371 tumors of 15 cancer types available in the TCGA, cancers where platinum constitutes standard firstline therapy showed increased genomic scar scores (61). Several combination HRD scores have been described (62), with most data for the 3-factor combination scar assay by Myriad Genetics (63). The performance of the combined TAI, LOH, and LST score to detect BRCA mutated cases, with minor adjustments to the definition criteria of the TAI and LST scores, was assessed in a cohort of 215 breast cancer tumors. All three individual scores—LOH, modified TAI, and LST—were statistically significantly associated with BRCA deficiency in all breast cancer samples. In a multivariable model, a combined mean HRD score was statistically significantly associated with BRCA deficiency, at an odds ratio (OR) of 87 (95% CI = 17 to 150). The combination score of LOH, TAI, and LST was also retrospectively validated to predict for response to neoadjuvant platinum therapy in three cohorts of triple-negative breast cancer (TNBC)—cisplatin-1, cisplatin-2, and PrECOG 0105 (64). Recently, the combined HRD score was also shown to predict pathologic complete response (pCR) after anthracycline- and/or taxane-based neoadjuvant chemotherapy in TNBC, irrespective of BRCA mutation status (65), as well as in TNBC patients after paclitaxel, doxorubicin, and bevacizumab with or without carboplatin (66). This combination score, marketed as the myChoice HRD test (Myriad Genetics), was further evaluated in the NOVA study of the PARP inhibitor niraparib in ovarian cancer (17). In the non-gBRCA mutated cohort of NOVA, a preplanned analysis on archival tumor tissue using the myChoice test showed a statistically significant benefit with niraparib treatment for myChoice-positive patients, which was greater than the benefit noted for non-HRD patients. High HRD score (>42) and/or BRCA1/2 mutation has also been shown to be associated with long-term response to olaparib (46). Among the single score assays, only the Foundation medicine LOH assay has been validated prospectively in the phase III setting (ARIEL 3 study) (Table 1). Table 1. Clinical evidence for regulatory approval of PARP inhibitors* Study/first author Design Cancer type Drug Inclusion Results Regulatory approval Kaufman et al. (14) Phase II (nonrandomized) Ovarian, breast, pancreatic, prostate Olaparib Deleterious gBRCA mutation and advanced solid tumor Overall response rate was 26%. Stable disease observed in 42%. FDA approval for maintenance treatment of gBRCA mutated recurrent ovarian cancer. Study 19 (15) Phase II (randomized, double-blind) Ovarian Olaparib Platinum-sensitive relapsed serous ovarian cancer Median PFS for olaparib vs placebo was 11.2 vs 4.3 months, respectively, in BRCA mutated patients and 7.5 vs 5.5 months, respectively, in BRCA wild-type. There was no OS benefit. EMA approval for maintenance in gBRCA mutated ovarian cancer. SOLO2/ENGOT-Ov21 (16) Phase III (randomized, double-blind) Ovarian Olaparib Relapsed ovarian cancer patients with a BRCA mutation, two prior lines of chemotherapy Median PFS for olaparib vs placebo was 19.1 vs 5.5 months, respectively. Additional FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. NOVA (17) Phase III (randomized, double-blind) Ovarian Niraparib Platinum-sensitive ovarian cancer Median PFS for olaparib vs placebo was 21.0 vs 5.5 months, respectively, in gBRCA mutated patients, 12.9 vs 3.8, respectively, in non-gBRCA mutated patients with HRD, and 9.3 vs 3.9 in the non-gBRCA mutated group without HRD. FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. Study 10 (18) Phase I–II (open-label) Ovarian Rucaparib Phase II enrolled platinum-sensitive gBRCA ovarian cancer, two to four prior lines of chemotherapy Objective response rate for gBRCA patients was 59.5%. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL2 (19) Phase II (open-label) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for BRCA mutated and LOH-high subgroups was 12.8 and 5.7 months, respectively, compared with 5.2 months in the LOH-low subgroup. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL3 (20) Phase III (randomized, double-blind) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for rucaparib vs placebo was 16.6 vs 5.4 months, respectively, in BRCA mutated patients and 13.6 vs 5.4, respectively, in the HRD group. FDA approval for advanced ovarian cancer. OlympiAD (21) Phase III (randomized, open label) Breast Olaparib HER2-negative metastatic breast cancer with gBRCA mutation Median PFS for olaparib vs chemotherapy (capecitabine, vinorelbine, eribulin) was 7.0 vs 4.2, respectively. Olaparib showed a higher response rate (59.9% vs 28.8%, olaparib and chemotherapy, respectively). FDA approval for metastatic breast cancer with gBRCA mutations. Study/first author Design Cancer type Drug Inclusion Results Regulatory approval Kaufman et al. (14) Phase II (nonrandomized) Ovarian, breast, pancreatic, prostate Olaparib Deleterious gBRCA mutation and advanced solid tumor Overall response rate was 26%. Stable disease observed in 42%. FDA approval for maintenance treatment of gBRCA mutated recurrent ovarian cancer. Study 19 (15) Phase II (randomized, double-blind) Ovarian Olaparib Platinum-sensitive relapsed serous ovarian cancer Median PFS for olaparib vs placebo was 11.2 vs 4.3 months, respectively, in BRCA mutated patients and 7.5 vs 5.5 months, respectively, in BRCA wild-type. There was no OS benefit. EMA approval for maintenance in gBRCA mutated ovarian cancer. SOLO2/ENGOT-Ov21 (16) Phase III (randomized, double-blind) Ovarian Olaparib Relapsed ovarian cancer patients with a BRCA mutation, two prior lines of chemotherapy Median PFS for olaparib vs placebo was 19.1 vs 5.5 months, respectively. Additional FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. NOVA (17) Phase III (randomized, double-blind) Ovarian Niraparib Platinum-sensitive ovarian cancer Median PFS for olaparib vs placebo was 21.0 vs 5.5 months, respectively, in gBRCA mutated patients, 12.9 vs 3.8, respectively, in non-gBRCA mutated patients with HRD, and 9.3 vs 3.9 in the non-gBRCA mutated group without HRD. FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. Study 10 (18) Phase I–II (open-label) Ovarian Rucaparib Phase II enrolled platinum-sensitive gBRCA ovarian cancer, two to four prior lines of chemotherapy Objective response rate for gBRCA patients was 59.5%. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL2 (19) Phase II (open-label) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for BRCA mutated and LOH-high subgroups was 12.8 and 5.7 months, respectively, compared with 5.2 months in the LOH-low subgroup. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL3 (20) Phase III (randomized, double-blind) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for rucaparib vs placebo was 16.6 vs 5.4 months, respectively, in BRCA mutated patients and 13.6 vs 5.4, respectively, in the HRD group. FDA approval for advanced ovarian cancer. OlympiAD (21) Phase III (randomized, open label) Breast Olaparib HER2-negative metastatic breast cancer with gBRCA mutation Median PFS for olaparib vs chemotherapy (capecitabine, vinorelbine, eribulin) was 7.0 vs 4.2, respectively. Olaparib showed a higher response rate (59.9% vs 28.8%, olaparib and chemotherapy, respectively). FDA approval for metastatic breast cancer with gBRCA mutations. * EMA = European Medicines Agency; FDA = Food and Drug Administration; gBRCA = germline BRCA1/2; HER2 = human epidermal growth factor receptor 2; HR = hazard ratio; HRD = homologous recombination deficiency; LOH = loss of heterozygosity; OS = overall survival; PARP = poly-ADP ribose polymerase; PFS = progression-free survival. Table 1. Clinical evidence for regulatory approval of PARP inhibitors* Study/first author Design Cancer type Drug Inclusion Results Regulatory approval Kaufman et al. (14) Phase II (nonrandomized) Ovarian, breast, pancreatic, prostate Olaparib Deleterious gBRCA mutation and advanced solid tumor Overall response rate was 26%. Stable disease observed in 42%. FDA approval for maintenance treatment of gBRCA mutated recurrent ovarian cancer. Study 19 (15) Phase II (randomized, double-blind) Ovarian Olaparib Platinum-sensitive relapsed serous ovarian cancer Median PFS for olaparib vs placebo was 11.2 vs 4.3 months, respectively, in BRCA mutated patients and 7.5 vs 5.5 months, respectively, in BRCA wild-type. There was no OS benefit. EMA approval for maintenance in gBRCA mutated ovarian cancer. SOLO2/ENGOT-Ov21 (16) Phase III (randomized, double-blind) Ovarian Olaparib Relapsed ovarian cancer patients with a BRCA mutation, two prior lines of chemotherapy Median PFS for olaparib vs placebo was 19.1 vs 5.5 months, respectively. Additional FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. NOVA (17) Phase III (randomized, double-blind) Ovarian Niraparib Platinum-sensitive ovarian cancer Median PFS for olaparib vs placebo was 21.0 vs 5.5 months, respectively, in gBRCA mutated patients, 12.9 vs 3.8, respectively, in non-gBRCA mutated patients with HRD, and 9.3 vs 3.9 in the non-gBRCA mutated group without HRD. FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. Study 10 (18) Phase I–II (open-label) Ovarian Rucaparib Phase II enrolled platinum-sensitive gBRCA ovarian cancer, two to four prior lines of chemotherapy Objective response rate for gBRCA patients was 59.5%. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL2 (19) Phase II (open-label) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for BRCA mutated and LOH-high subgroups was 12.8 and 5.7 months, respectively, compared with 5.2 months in the LOH-low subgroup. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL3 (20) Phase III (randomized, double-blind) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for rucaparib vs placebo was 16.6 vs 5.4 months, respectively, in BRCA mutated patients and 13.6 vs 5.4, respectively, in the HRD group. FDA approval for advanced ovarian cancer. OlympiAD (21) Phase III (randomized, open label) Breast Olaparib HER2-negative metastatic breast cancer with gBRCA mutation Median PFS for olaparib vs chemotherapy (capecitabine, vinorelbine, eribulin) was 7.0 vs 4.2, respectively. Olaparib showed a higher response rate (59.9% vs 28.8%, olaparib and chemotherapy, respectively). FDA approval for metastatic breast cancer with gBRCA mutations. Study/first author Design Cancer type Drug Inclusion Results Regulatory approval Kaufman et al. (14) Phase II (nonrandomized) Ovarian, breast, pancreatic, prostate Olaparib Deleterious gBRCA mutation and advanced solid tumor Overall response rate was 26%. Stable disease observed in 42%. FDA approval for maintenance treatment of gBRCA mutated recurrent ovarian cancer. Study 19 (15) Phase II (randomized, double-blind) Ovarian Olaparib Platinum-sensitive relapsed serous ovarian cancer Median PFS for olaparib vs placebo was 11.2 vs 4.3 months, respectively, in BRCA mutated patients and 7.5 vs 5.5 months, respectively, in BRCA wild-type. There was no OS benefit. EMA approval for maintenance in gBRCA mutated ovarian cancer. SOLO2/ENGOT-Ov21 (16) Phase III (randomized, double-blind) Ovarian Olaparib Relapsed ovarian cancer patients with a BRCA mutation, two prior lines of chemotherapy Median PFS for olaparib vs placebo was 19.1 vs 5.5 months, respectively. Additional FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. NOVA (17) Phase III (randomized, double-blind) Ovarian Niraparib Platinum-sensitive ovarian cancer Median PFS for olaparib vs placebo was 21.0 vs 5.5 months, respectively, in gBRCA mutated patients, 12.9 vs 3.8, respectively, in non-gBRCA mutated patients with HRD, and 9.3 vs 3.9 in the non-gBRCA mutated group without HRD. FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. Study 10 (18) Phase I–II (open-label) Ovarian Rucaparib Phase II enrolled platinum-sensitive gBRCA ovarian cancer, two to four prior lines of chemotherapy Objective response rate for gBRCA patients was 59.5%. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL2 (19) Phase II (open-label) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for BRCA mutated and LOH-high subgroups was 12.8 and 5.7 months, respectively, compared with 5.2 months in the LOH-low subgroup. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL3 (20) Phase III (randomized, double-blind) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for rucaparib vs placebo was 16.6 vs 5.4 months, respectively, in BRCA mutated patients and 13.6 vs 5.4, respectively, in the HRD group. FDA approval for advanced ovarian cancer. OlympiAD (21) Phase III (randomized, open label) Breast Olaparib HER2-negative metastatic breast cancer with gBRCA mutation Median PFS for olaparib vs chemotherapy (capecitabine, vinorelbine, eribulin) was 7.0 vs 4.2, respectively. Olaparib showed a higher response rate (59.9% vs 28.8%, olaparib and chemotherapy, respectively). FDA approval for metastatic breast cancer with gBRCA mutations. * EMA = European Medicines Agency; FDA = Food and Drug Administration; gBRCA = germline BRCA1/2; HER2 = human epidermal growth factor receptor 2; HR = hazard ratio; HRD = homologous recombination deficiency; LOH = loss of heterozygosity; OS = overall survival; PARP = poly-ADP ribose polymerase; PFS = progression-free survival. Table 3. SNP-based “genomic scar” assays* Assay Description Biological rationale Retrospective validation Prospective validation Telomeric allelic imbalance Subchromosomal regions displaying allelic imbalance extending to the telomere but not crossing the centromere (58) HRD leads to aberrant chromosomal end fusions due to inappropriate end joining, which would then lead to an allelic-imbalance after breakage during mitosis. Neoadjuvant cisplatin for TNBC showed a statistically significant correlation between higher levels of TAI and response. TCGA data show elevated TAI in gBRCA mutated ovarian cancer (58). Not as single assay (combination studies only) Loss of heterozygosity Analysis of the number of regions with LOH, of more than 15 Mb and shorter than the whole chromosome (59) HRD results in uniparental disomy or LOH due to inaccurate repair of sister chromatids during the S/G2 phase of the cell cycle. Higher levels of LOH are seen in BRCA-deficient ovarian cancers than in sporadic cancers (59,67) and are associated with lower chemotherapy resistance rates and longer PFS (68). In the ARIEL2 study, there was a longer PFS in the LOH-high subgroup compared with LOH-low (HR= 0.62, 95% CI = 0.42 to 0.90) (19). Large-scale transition Chromosomal breaks of more than 10 Mb (after exclusion of regions shorter than 3 Mb) (60) Large structural chromosomal aberrations arise in HRD cells due to inappropriate recombination between chromosomal segments. BRCA/RAD51C-inactivated breast cancers show higher LST than sporadic cancers (60,69). Not as single assay (combination studies only) Assay Description Biological rationale Retrospective validation Prospective validation Telomeric allelic imbalance Subchromosomal regions displaying allelic imbalance extending to the telomere but not crossing the centromere (58) HRD leads to aberrant chromosomal end fusions due to inappropriate end joining, which would then lead to an allelic-imbalance after breakage during mitosis. Neoadjuvant cisplatin for TNBC showed a statistically significant correlation between higher levels of TAI and response. TCGA data show elevated TAI in gBRCA mutated ovarian cancer (58). Not as single assay (combination studies only) Loss of heterozygosity Analysis of the number of regions with LOH, of more than 15 Mb and shorter than the whole chromosome (59) HRD results in uniparental disomy or LOH due to inaccurate repair of sister chromatids during the S/G2 phase of the cell cycle. Higher levels of LOH are seen in BRCA-deficient ovarian cancers than in sporadic cancers (59,67) and are associated with lower chemotherapy resistance rates and longer PFS (68). In the ARIEL2 study, there was a longer PFS in the LOH-high subgroup compared with LOH-low (HR= 0.62, 95% CI = 0.42 to 0.90) (19). Large-scale transition Chromosomal breaks of more than 10 Mb (after exclusion of regions shorter than 3 Mb) (60) Large structural chromosomal aberrations arise in HRD cells due to inappropriate recombination between chromosomal segments. BRCA/RAD51C-inactivated breast cancers show higher LST than sporadic cancers (60,69). Not as single assay (combination studies only) * CI = confidence interval; gBRCA = germline BRCA1/2; HR = hazard ratio; HRD = homologous recombination deficiency; LOH = loss of heterozygosity; LST = large-scale transition; PFS = progression-free survival; SNP = single nucleotide polymorphism; TAI = telomeric allelic imbalance; TCGA = The Cancer Genome Atlas; TNBC = triple-negative breast cancer Table 3. SNP-based “genomic scar” assays* Assay Description Biological rationale Retrospective validation Prospective validation Telomeric allelic imbalance Subchromosomal regions displaying allelic imbalance extending to the telomere but not crossing the centromere (58) HRD leads to aberrant chromosomal end fusions due to inappropriate end joining, which would then lead to an allelic-imbalance after breakage during mitosis. Neoadjuvant cisplatin for TNBC showed a statistically significant correlation between higher levels of TAI and response. TCGA data show elevated TAI in gBRCA mutated ovarian cancer (58). Not as single assay (combination studies only) Loss of heterozygosity Analysis of the number of regions with LOH, of more than 15 Mb and shorter than the whole chromosome (59) HRD results in uniparental disomy or LOH due to inaccurate repair of sister chromatids during the S/G2 phase of the cell cycle. Higher levels of LOH are seen in BRCA-deficient ovarian cancers than in sporadic cancers (59,67) and are associated with lower chemotherapy resistance rates and longer PFS (68). In the ARIEL2 study, there was a longer PFS in the LOH-high subgroup compared with LOH-low (HR= 0.62, 95% CI = 0.42 to 0.90) (19). Large-scale transition Chromosomal breaks of more than 10 Mb (after exclusion of regions shorter than 3 Mb) (60) Large structural chromosomal aberrations arise in HRD cells due to inappropriate recombination between chromosomal segments. BRCA/RAD51C-inactivated breast cancers show higher LST than sporadic cancers (60,69). Not as single assay (combination studies only) Assay Description Biological rationale Retrospective validation Prospective validation Telomeric allelic imbalance Subchromosomal regions displaying allelic imbalance extending to the telomere but not crossing the centromere (58) HRD leads to aberrant chromosomal end fusions due to inappropriate end joining, which would then lead to an allelic-imbalance after breakage during mitosis. Neoadjuvant cisplatin for TNBC showed a statistically significant correlation between higher levels of TAI and response. TCGA data show elevated TAI in gBRCA mutated ovarian cancer (58). Not as single assay (combination studies only) Loss of heterozygosity Analysis of the number of regions with LOH, of more than 15 Mb and shorter than the whole chromosome (59) HRD results in uniparental disomy or LOH due to inaccurate repair of sister chromatids during the S/G2 phase of the cell cycle. Higher levels of LOH are seen in BRCA-deficient ovarian cancers than in sporadic cancers (59,67) and are associated with lower chemotherapy resistance rates and longer PFS (68). In the ARIEL2 study, there was a longer PFS in the LOH-high subgroup compared with LOH-low (HR= 0.62, 95% CI = 0.42 to 0.90) (19). Large-scale transition Chromosomal breaks of more than 10 Mb (after exclusion of regions shorter than 3 Mb) (60) Large structural chromosomal aberrations arise in HRD cells due to inappropriate recombination between chromosomal segments. BRCA/RAD51C-inactivated breast cancers show higher LST than sporadic cancers (60,69). Not as single assay (combination studies only) * CI = confidence interval; gBRCA = germline BRCA1/2; HR = hazard ratio; HRD = homologous recombination deficiency; LOH = loss of heterozygosity; LST = large-scale transition; PFS = progression-free survival; SNP = single nucleotide polymorphism; TAI = telomeric allelic imbalance; TCGA = The Cancer Genome Atlas; TNBC = triple-negative breast cancer Mutational Signatures Cancer types bear distinct mutational signatures, that is, mutational patterns (as opposed to isolated detection of mutations at selected loci), likely acquired through distinct patterns of mutagen exposure and genomic instability, which can be identified using NGS. The overall genome-wide mutation burden is associated with improved survival after chemotherapy in BRCA mutated ovarian cancers (70). Interestingly, BRCA2 mutated ovarian cancers are associated with a higher mutator phenotype than BRCA1 mutant cancers (71). This underscores a need for multidimensional scores to unravel the complexity of HRD patterns. The mutational burden assay was refined by combining somatic mutation count with copy number variations to improve the identification of BRCA mutated ovarian cancer patients from the TCGA (72). The incorporation of pattern of mutations, in addition to mutational burden, has led to the development of specific “signatures” of mutations in cancer, based on base substitution patterns. One of these, “signature 3,” described by Alexandrov et al., is attributed to underlying HRD (73) and has been shown to exist in several cancers, including breast, ovarian, prostate, and gastric. HRDetect, a weighted model of mutational signatures, has recently demonstrated good performance in predicting BRCA deficiency (defined as somatic or germline mutations and BRCA1 promoter hypermethylation) in breast, ovarian, and pancreatic cancers (74). This model incorporates a weighted score of microhomology-mediated deletions, base substitution/rearrangement signatures, and the HRD index based on genomic scars. HRDetect identifies BRCA-deficient tumors at high sensitivity and specificity from FFPE samples (area under the receiver operating characteristic curve = 0.98). Additionally, one-third of the HRD-high score cases were BRCA wild-type and did not associate with known mutations in other homologous recombination genes. However, the HRDetect algorithm has yet to be validated in a clinical trial of PARP inhibition. Limitations of Genomic Scar Assays Limitations of the genomic scar assays were highlighted by the inability of the Myriad myChoice assay to predict for platinum sensitivity in the TNT study of docetaxel or carboplatin in metastatic TNBC patients (32). Prior anthracycline exposure in this setting may contribute to the inaccuracy of the assay, as the emergence of resistance pathways would not remove the existing “scar” of prior HRD. This is substantiated by the improved performance of this assay in the neoadjuvant setting (64). The predictive potential of genomic scar HRD assays could therefore be undermined by mechanisms dynamically affecting the homologous recombination pathway and drug accumulation. BRCA1/2 (30,75), and recently RAD51C/D (76) and PALB2 (77), reversions or secondary mutations have been described to restore homologous recombination. A BRCA mutation may have initially imprinted a genomic HRD scar signature, but, upon reversion, the tumor would regain homologous recombination functionality even if the HRD scar was still detectable. This is particularly relevant in ovarian cancer, where approximately half of all platinum-resistant BRCA mutated tumors eventually develop restoration of BRCA function in response to platinum therapy (78,79). Restoration of BRCA2 function was also reported in pancreatic cancer in response to the PARP inhibitor olaparib (80). Other BRCA-dependent resistance mechanisms include stabilization of BRCT domain mutants of BRCA1 by HSP90 (81), alternative splicing (82), or alternative translation initiation of BRCA1 (83). BRCA1-independent mechanisms governing PARP inhibitor resistance leverage on promoting genomic stability at stalled replication forks (84,85) or modulation of double-strand DNA break end resection (86,87). Finally, acquired or intrinsic resistance is frequently mediated by membrane transporters (88), as described in olaparib resistance in mouse models due to upregulation of the efflux pump P-glycoprotein (89). These mechanisms are not captured in genomic scar HRD assays. A negative myChoice HRD scar assay does not also exclude HRD. In the phase III NOVA clinical trial of maintenance therapy of PARP inhibitor niraparib in platinum-sensitive recurrent ovarian cancer (17), niraparib improved progression-free survival (PFS), even in the cohort classified as HRD proficient (6.9 vs 3.8 months; HR = 0.58, 95% CI = 0.36 to 0.92). NOVA highlights challenges with the use of SNP-based genomic scar assays to exclude patients for PARP inhibitor therapy, especially in a platinum-sensitive setting. For the caveats listed above, assays that report a “real-time” index of HR in the tumor sample would be of clinical interest and value. Real-time Indicators of HRD Unlike genomic alterations, which are reflective of past events, RNA and proteins vary dynamically in quantity and localization during cellular processes. They are therefore postulated to provide a current snapshot of the state of HRD in a given tumor sample. Transcriptional Profiles Gene expression profiling (GEP) captures a current transcriptional state of a tumor, with platforms such as OncotypeDx already in clinical use for the selection of patients for adjuvant chemotherapy (90). GEP assays, including the 93-gene Chemotherapy Response Profile (CRP), have similarly been retrospectively evaluated, with promising results in the context of platinum responses in ovarian cancer (91–93). These GEP panels, though not exclusively focused on homologous recombination, have interrogated genes that regulate apoptosis, cell cycle entry, and DNA repair. BRCA germline mutated ovarian tumors show a distinct GEP from sporadic tumors and normal ovarian surface epithelium (94). BRCA profiles are present in some sporadic tumors (95), however, highlighting the potential value of such GEP assays as a broader net for HRD in cancer. A 60-gene signature clusters tumors into BRCA-like and non-BRCA-like (96), and this BRCAness profile could predict platinum sensitivity even within a BRCA mutant population. Importantly, the development of platinum resistance during the course of therapy was reflected in the change of BRCAness profile in two out of four patients, highlighting the dynamic nature of these assays. In breast cancer, an FFPE-compatible 44-gene microarray-based assay, termed the DDR deficiency assay (97), predicted complete pathologic response after neoadjuvant chemotherapy (OR = 4.0) and improved five-year relapse-free survival (HR = 0.37, 95% CI = 0.15 to 0.88). A 77-gene expression profile termed BRCA1ness was developed based on a cohort of TNBC associated with a known BRCA1-like aCGH profile (55,98). Genes included in this profile constitute signatures of DNA replication, recombination and repair, cellular function/maintenance, cellular assembly, and cell cycle, and also metabolic signatures of serine, glycine, and histamine biosynthesis. The BRCA1ness GEP was evaluated in the phase II I-SPY 2 study, where human epidermal growth factor receptor 2–negative breast cancer patients were randomly assigned to receive paclitaxel with or without concurrent veliparib and carboplatin (V-C). Patients with a BRCA1ness GEP showed a statistically significantly higher proportion of pCR at an odds ratio of 3.2 only in the V-C treated group. At present, none of the above GEP assays have been specifically assessed in the context of PARP inhibition. Their comparative performance in retrospective clinical trial cohorts and compatibility with FFPE samples will be critical in further development. Protein Expression The expression of tumor suppressor proteins involved in homologous recombination has been analyzed in cancer tissue using immunohistochemical approaches, typically in retrospective studies. Immunohistochemical analyses of the BRCA1 and 2 proteins have been hindered by the absence of validated reagents and low expression of these proteins. Among non-BRCA DDR proteins, ataxia-telangiectasia mutated kinase (ATM) has been evaluated most extensively in clinical studies of PARP inhibition. ATM operates upstream of homologous recombination in the DNA damage response, and depletion of this protein is associated with PARP inhibitor sensitivity in vitro (38,99–101). ATM loss as measured by immunohistochemistry (IHC) has been described in various tumor groups, including gastric (102), colon (103), and breast (104) cancer. In gastric cancer, ATM loss occurs in about 20% of the population (99,102). A randomized phase II study of paclitaxel+olaparib compared with paclitaxel+placebo in gastric cancer demonstrated OS benefit in the ATM loss population (105). Unfortunately, this was not borne out in the follow-up randomized phase III GOLD study (106). The loss of ATM can be partially compensated for by other DNA repair pathways, which were not measured in the GOLD trial samples. Single protein IHC assays are likely to be superseded by novel approaches, such as multispectral microscopy, tissue mass cytometry, and digital spatial profiling, which provide multiplexed information in the histological analysis of proteins. The application of multiplex analyses of DNA repair proteins in clinical trial material may herald new insights into biomarkers of HRD. Functional Assays Phenotypic or functional assays are appealing, given the challenges of measuring all proteins of interest within a pathway for each cancer sample. For example, a functional assay of HRD should ideally measure a single downstream event that would reflect proficiency of multiple upstream components of homologous recombination. Quantification of RAD51 foci is a prototypical example of a functional HRD assay. In the S/G2 phases of the cell cycle, when the sister chromatid is available for recombination to occur, RAD51 forms distinct subnuclear foci after DNA-damaging insults. The inability of cells to form RAD51 foci is a common feature of HRD. It potentially provides a global read-out of HRD without defining the underlying cause of homologous recombination deficiency or reversion, as many of the factors influencing these might still be unknown. RAD51 foci in S/G2 cells were evaluated in FFPE breast cancer tissue 24 hours postchemotherapy (107) using a counterstain to geminin, a marker for S and G2 cells (108). RAD51-low score was more common in TNBC than in other subtypes, with higher pCR rates (33% vs 3%). A major challenge to be overcome is the reliable quantitation of “foci” in FFPE samples, where fixation artefacts are common. Furthermore, the baseline level of RAD51 (and other DDR) foci is not as relevant as the relative increase in their numbers upon DNA damage. Therefore, another challenge with such assays is the lack of availability of post-treatment biopsies in routine clinical practice. Ex vivo experiments offer a workaround, for example, in the investigation of ionizing radiation–induced RAD51 foci in organotypic breast cancer samples (109) or primary cultures of ascites in epithelial ovarian cancer patients with rucaparib treatment ex vivo (110). While these studies point to the potential of RAD51 as a composite marker of homologous recombination, several technical and practical challenges curtail its clinical utility in its current form. The tissue context, modality of DNA damage used, timing of RAD51 foci assessment (111), and resolution of the microscopy system all account for the large differences of foci assessment between these studies. False-negative RAD51 foci formation assays may also occur with defects operating downstream of RAD51-ssDNA filament formation, such as depletion of RAD51-associated protein 1 (RAD51AP1) (112) and polymerase eta disruption (113). Cells with loss of the upstream DDR components of the MRN complex (114) and ATM form RAD51 foci in vitro, but display defects in homologous recombination (115,116). Finally, resistance to PARP inhibition in mouse models is associated with a persistent RAD51 focus formation defect, which points to a critical role for replication fork stabilization in PARP inhibitor sensitivity (84). Therefore, at present, RAD51 foci formation remains a biologically useful marker that is not easily quantifiable in clinical material. A clinically measurable composite marker of replication fork stabilization may, however, offer a promising phenotypic marker of PARP inhibitor sensitivity. Conclusions HRD is a bona fide anticancer target where multiple therapeutic modalities have demonstrated varying degrees of efficacy, including chemotherapy and DNA repair enzyme inhibition. The aforementioned biomarkers have provided tantalizing hints of their relevance in specific tumor types. Nonetheless, the development of a generic touchstone biomarker for HRD will be key to expanding the therapeutic utility of HRD-targeting agents across a broad spectrum of tumor types. Given the complexity of the homologous recombination pathway, it is unlikely that one single biomarker will suffice. From a practical standpoint, clinical surrogate biomarkers like platinum sensitivity are likely to prevail for the foreseeable future, although these are neither ideal nor sufficiently comprehensive. Primary platinum resistance, for example, may be mediated by mechanisms that are independent of homologous recombination status, and the inclusion of only platinum-sensitive patients will deny a subgroup of homologous recombination-deficient patients the possible benefits of PARP inhibition (Figure 1A). In all likelihood, composite HRD scores involving two or more biomarker strategies will be eventually be required to define “HRDness.” An example for such a score could be “platinum sensitivity OR HRDetect positivity AND decreased replication fork stability.” Such techniques will only be suitable for clinical application if they can provide rapid and precise readouts. The nature of the genomic instability in HRD tumor cells also confers accelerated capacity for therapeutic evasion via clonal evolution. As such, a cornerstone of success in targeting HRD will be to ensure that the optimal therapeutic strategy is applied as early as possible in the course of a patient’s treatment. The difficulties of validating any biomarker approach will be compounded by tumor heterogeneity and clonal evolution in late stages of the disease and treatment. Consequently, this implies a need for constant re-evaluation of tumor homologous recombination status in patients for whom HRD-targeted drugs are being considered. In this context, HRD biomarkers based on tissue biopsy techniques that enable serial and multiple spatially distinct samples or liquid biopsies may be the key to surmounting these hurdles. Ultimately, overcoming challenges of identifying the right patients at the right time for the right therapeutic strategy will allow the full potential of HRD-directed precision medicine to be realized. Funding ADJ and DSPT are recipients of the National Medical Research Council Transition Award and the Clinician-Scientist Award, respectively. RS is supported by the MOH Healthcare Research Scholarship. MMH is a recipient of the Cancer Science Institute of Singapore PhD Graduate Scholarship. This work is supported by the Singapore Ministry of Health's National Medical Research Council under a clinician-scientist grant to DSPT (NMRC/CIRG/1400/2014). Notes Affiliations of authors: Cancer Science Institute of Singapore (MMH, DSPT, ADJ) and Department of Haematology-Oncology (RS, DSPT, ADJ), National University Hospital, Singapore. The funders had no role in the writing of this Commentary or the decision to submit it for publication. The authors wish to thank Celestina Chin for proofreading the manuscript, Piotr Garstecki for help with editing of figures, and Prof. Stanley Kaye for his helpful discussions and insight. Conflicts of interest: ADJ: honoraria from MSD; DSPT: honoraria from AstraZeneca, Roche, and MSD and research funding from AstraZeneca, Bayer, and Karyopharm. References 1 Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer . 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Biomarkers for Homologous Recombination Deficiency in Cancer

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Abstract

Abstract Defective DNA repair is a common hallmark of cancer. Homologous recombination is a DNA repair pathway of clinical interest due to the sensitivity of homologous recombination-deficient cells to poly-ADP ribose polymerase (PARP) inhibitors. The measurement of homologous recombination deficiency (HRD) in cancer is therefore vital to the appropriate design of clinical trials incorporating PARP inhibitors. However, methods to identify HRD in tumors are varied and controversial. Understanding existing and new methods to measure HRD is important to their appropriate use in clinical trials and practice. The aim of this review is to summarize the biology and clinical validation of current methods to measure HRD, to aid decision-making for patient stratification and translational research in PARP inhibitor trials. We discuss the current clinical development of PARP inhibitors, along with established indicators for HRD such as germline BRCA1/2 mutation status and clinical response to platinum-based therapy. We then examine newer assays undergoing clinical validation, including 1) somatic mutations in homologous recombination genes, 2) “genomic scar” assays using array-based comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) analysis or mutational signatures derived from next-generation sequencing, 3) transcriptional profiles of HRD, and 4) phenotypic or functional assays of protein expression and localization. We highlight the strengths and weaknesses of each of these assays, for consideration during the design of studies involving PARP inhibitors. Each cell is equipped with a versatile set of DNA damage response (DDR) networks that guard the genome against mutational insults (1). Double-strand breaks (DSBs) are a particularly hazardous form of DNA damage and are repaired by two major repair pathways—error-free homologous recombination and nonhomologous end-joining (NHEJ) (2,3). Homologous recombination deficiency (HRD) was initially described in cancers with germline mutations of the tumor suppressors BRCA1 and BRCA2 (4). However, genetic and epigenetic inactivation of other homologous recombination components can lead to HRD in sporadic cancers, broadly termed BRCAness (5–7). As homologous recombination is required for the repair of DSBs generated during DNA interstrand cross-link (ICL) resolution (8), homologous recombination-deficient tumors are sensitive to ICL-generating platinum chemotherapy (9). Furthermore, inhibitors of the DNA repair enzyme poly-ADP ribose polymerase 1 (PARP1) (10) have rapidly gained clinical interest following observations that cells with mutant BRCA1 and BRCA2 display exquisite sensitivity to these agents (11,12). This sensitivity of BRCA1/2 mutant cells to PARP inhibition results from the synthetic lethality of cells with defective homologous recombination-mediated DNA repair to toxic replication intermediates generated on chromatin by PARP “trapping” (13). Three PARP inhibitors (olaparib, rucaparib, and niraparib) are currently approved by the Food and Drug Administration (FDA) (Table 1). Biomarkers that predict HRD in BRCA wild-type tumors could theoretically expand the use of PARP inhibitors beyond BRCA mutant tumors. Here we aim to summarize these markers in the context of their biology, clinical validation, and potential clinical utility in trials targeting HRD. Approved HRD Biomarkers for PARP Inhibitor Use Germline BRCA Mutations The clinical evidence so far suggests that germline BRCA (gBRCA) mutations remain the best clinical biomarkers for response to PARP inhibitor therapy (17,21,22), and BRCA mutant cells show clear evidence of HRD in vitro (23,24). PARP inhibitor trials have used distinct sequencing assays to evaluate the presence of gBRCA mutations (25–28). The Myriad Genetics BRACAnalysis CDx platform (Myriad Genetics; Salt Lake City, UT) has been FDA approved to identify ovarian cancer patients with suspected deleterious gBRCA variants eligible for treatment with olaparib. This blood-based assay was validated retrospectively and matched to local results via a bridging study (29). Larger phase III studies of PARP inhibitors in ovarian cancer (study 19 [15] and the NOVA trial [17]) and breast cancer (OlympiAD [21]) subsequently used BRACAnalysis to establish gBRCA mutation status. There are currently no approved diagnostic assays for HRD based on germline mutations of other homologous recombination genes. Using germline mutations in homologous recombination genes to classify tumors as HRD, however, suffers from several drawbacks. Variants of uncertain significance (VUS) remain a problem, as they do for germline testing of tumor suppressor genes toward familial risk prediction, as the genotype-phenotype correlations of many variants are unclear. Somatic reversion mutations may render a tumor functionally homologous recombination proficient even with germline mutations in homologous recombination repair–related genes (30). Ideally, biomarkers of HRD should not only predict HRD in BRCA wild-type tumors, but also refine the BRCA mutant population to account for phenotypic variants and reversions. The absence of BRCA locus–specific LOH, for instance, is associated with poorer platinum response in gBRCA mutant tumors (31). Because of these caveats, there have been efforts to generate surrogate biomarkers of homologous recombination for patient stratification in PARP inhibitor trials. Platinum Sensitivity as a Surrogate Biomarker for HRD Platinum sensitivity in vitro is a feature of homologous recombination-deficient cells, and BRCA mutant ovarian and breast tumors demonstrate increased platinum sensitivity (9,32). As platinum is a key component of firstline chemotherapy in ovarian cancer, prior platinum sensitivity has been evaluated as a surrogate clinical index for prediction of efficacy to PARP inhibition (26). In the phase III NOVA study (17) of niraparib in platinum-sensitive ovarian cancer, the highest benefit was seen in gBRCA mutated patients. However, there was a benefit noted in all subsets of platinum-sensitive ovarian cancer (17), leading to niraparib’s approval for all platinum-sensitive metastatic ovarian cancers. PARP inhibitor sensitivity does not show complete overlap with platinum sensitivity (22). For example, cancers with defects in nucleotide excision repair (NER) may respond to platinum therapy, though this does not confer a concurrent PARP inhibitor sensitivity (33). Conversely, there is also a proportion of platinum-resistant patients who remain sensitive to PARP inhibitors, which would be missed by a “platinum-sensitive only” indication (Figure 1A) (14). For these reasons, it remains pertinent to explore assays that may accurately define HRD status in tumors. Novel Biomarkers of HRD A plethora of markers have been described to confer PARP inhibitor sensitivity in vitro (34,35), but most of these have not been validated clinically in well-annotated data sets. Here we categorize the various methods utilized for HRD diagnosis to date and review their biological and clinical rationale (Figure 1B). Figure 1. View largeDownload slide Homologous recombination deficiency (HRD) in cancers and assays for its estimation. A) The relationship between HRD, platinum, and poly-ADP ribose polymerase (PARP) inhibitor sensitivity in cancer. Most HRD tumors are sensitive to both platinum and PARP inhibition, but these sets do not overlap completely. Most PARP inhibitor–sensitive tumors will be HRD, but some HRD tumors will be PARP inhibitor resistant. HRD tumors may be platinum resistant due to drug efflux pumps and yet retain PARP inhibitor sensitivity. Conversely, tumors may also be platinum sensitive due to defects in nucleotide excision repair, which does not confer synthetic lethality to PARP inhibitors. B) An overview of HRD assays and their biological rationale. Key proteins involved in homologous recombination are depicted in the upper left section, and the color code depicts the nature of their typical alteration in cancer (red: somatic mutations; blue: germline mutations; green: altered gene expression; for clarity, only the most prevalent alteration is color-coded). These alterations lead to functional homologous recombination deficiency, which is reported by downstream assays of homologous recombination. The impact of HRD is the accumulation of specific patterns of mutations across the genome, which are studied using genomic scar assays. DSB = double-strand DNA breaks; HR = homologous recombination; LOH = loss of heterozygosity; LST = large-scale transition; TAI = telomeric allelic imbalance. Figure 1. View largeDownload slide Homologous recombination deficiency (HRD) in cancers and assays for its estimation. A) The relationship between HRD, platinum, and poly-ADP ribose polymerase (PARP) inhibitor sensitivity in cancer. Most HRD tumors are sensitive to both platinum and PARP inhibition, but these sets do not overlap completely. Most PARP inhibitor–sensitive tumors will be HRD, but some HRD tumors will be PARP inhibitor resistant. HRD tumors may be platinum resistant due to drug efflux pumps and yet retain PARP inhibitor sensitivity. Conversely, tumors may also be platinum sensitive due to defects in nucleotide excision repair, which does not confer synthetic lethality to PARP inhibitors. B) An overview of HRD assays and their biological rationale. Key proteins involved in homologous recombination are depicted in the upper left section, and the color code depicts the nature of their typical alteration in cancer (red: somatic mutations; blue: germline mutations; green: altered gene expression; for clarity, only the most prevalent alteration is color-coded). These alterations lead to functional homologous recombination deficiency, which is reported by downstream assays of homologous recombination. The impact of HRD is the accumulation of specific patterns of mutations across the genome, which are studied using genomic scar assays. DSB = double-strand DNA breaks; HR = homologous recombination; LOH = loss of heterozygosity; LST = large-scale transition; TAI = telomeric allelic imbalance. Somatic Mutations in Homologous Recombination Genes Somatic mutations in BRCA genes are more common than gBRCA mutations. In high-grade serous ovarian cancer, approximately 7% of patients have BRCA somatic mutations and approximately 14% have germline mutations (36,37). The tissue-based FoundationFocus CDxBRCA assay (Foundation medicine; Cambridge, MA) examines both germline and somatic mutations in the tumor and is FDA approved as a companion diagnostic to rucaparib based on the ARIEL trials (19,20). In vitro studies show that determinants of PARP inhibitor sensitivity beyond BRCA1 and BRCA2 include proteins involved in homologous recombination and related pathways (38). In vitro criteria that predict sensitivity to PARP inhibitors include 1) defects in recombination substrate assays (39); 2) inability for RAD51 to form distinct nuclear foci required for functional homologous recombination upon exposure to DNA damage (40); and 3) in vitro sensitivity to platinum salts and PARP inhibitors (11). Cancer-associated mutations in genes fulfilling the above criteria, including PALB2, BARD1, BRIP1, RAD51B, RAD51C, RAD51D, ATM, FAAP20, CHEK2, FAN1, FANCE, FANCM, and POLQ (36,41), are potential biomarkers of HRD in cancer but suffer from the same uncertainty over functional significance as germline mutations. Nonetheless, several of these mutations correlate with clinical responses to PARP inhibitor treatment (Table 2), with the TOPARP study (42) in prostate cancer being a notable example. Apart from mutational inactivation, BRCA1 can also be suppressed through promoter methylation. Notably however, BRCA1-methylated ovarian cancer patients do not appear to have a survival benefit with platinum-based chemotherapy (36,43–45) or demonstrate long-term response to PARP inhibitor therapy (46). Table 2. Clinical evidence of PARP inhibitor sensitivity to somatic mutations in homologous recombination genes* Study Design Cancer type Drug Platform to detect mutations Homologous recombination genes studied Results TOPARP (Mateo et al., 2015) (42) Single-arm phase II Prostate Olaparib GeneRead DNAseq Panel (Qiagen, Germany) BRCA1, BRCA2, ATM, FANCA, CHEK2, PALB2, NBN, HDAC2 16 of 50 patients (33%) had tumor aberrations in DNA-repair genes, with 14 of 16 biomarker-positive patients (88%) having a response to olaparib. ARIEL2 (Swisher et al., 2017) (19) Single-arm phase II Ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) BRCA1, BRCA2, ATM, BRIP1, CHEK2, FANCA, FANCI, FANCM, NBN, RAD51C, RAD51D, RAD54L 74% of patients with somatic BRCA mutations had an objective response. Response was also noted in patients with mutations in non-BRCA homologous recombination genes (ATM, NBN, RAD51C, and RAD51D). ARIEL3 (Coleman et al., 2017) (20) Phase III High-grade serous ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) (somatic) BRCA BRCA mutated patients had longer PFS compared with the placebo group (13.6 vs 5.4 months, HR = 0.32, 95% CI = 0.24 to 0.42). BRCAnalysis CDx test (Myriad Genetics, Salt Lake City, UT) (germline) (Multiple other genes on NGS) Study Design Cancer type Drug Platform to detect mutations Homologous recombination genes studied Results TOPARP (Mateo et al., 2015) (42) Single-arm phase II Prostate Olaparib GeneRead DNAseq Panel (Qiagen, Germany) BRCA1, BRCA2, ATM, FANCA, CHEK2, PALB2, NBN, HDAC2 16 of 50 patients (33%) had tumor aberrations in DNA-repair genes, with 14 of 16 biomarker-positive patients (88%) having a response to olaparib. ARIEL2 (Swisher et al., 2017) (19) Single-arm phase II Ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) BRCA1, BRCA2, ATM, BRIP1, CHEK2, FANCA, FANCI, FANCM, NBN, RAD51C, RAD51D, RAD54L 74% of patients with somatic BRCA mutations had an objective response. Response was also noted in patients with mutations in non-BRCA homologous recombination genes (ATM, NBN, RAD51C, and RAD51D). ARIEL3 (Coleman et al., 2017) (20) Phase III High-grade serous ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) (somatic) BRCA BRCA mutated patients had longer PFS compared with the placebo group (13.6 vs 5.4 months, HR = 0.32, 95% CI = 0.24 to 0.42). BRCAnalysis CDx test (Myriad Genetics, Salt Lake City, UT) (germline) (Multiple other genes on NGS) * CI = confidence interval; HR = hazard ratio; NGS = next-generation sequencing; PARP = poly-ADP ribose polymerase; PFS = progression-free survival. Table 2. Clinical evidence of PARP inhibitor sensitivity to somatic mutations in homologous recombination genes* Study Design Cancer type Drug Platform to detect mutations Homologous recombination genes studied Results TOPARP (Mateo et al., 2015) (42) Single-arm phase II Prostate Olaparib GeneRead DNAseq Panel (Qiagen, Germany) BRCA1, BRCA2, ATM, FANCA, CHEK2, PALB2, NBN, HDAC2 16 of 50 patients (33%) had tumor aberrations in DNA-repair genes, with 14 of 16 biomarker-positive patients (88%) having a response to olaparib. ARIEL2 (Swisher et al., 2017) (19) Single-arm phase II Ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) BRCA1, BRCA2, ATM, BRIP1, CHEK2, FANCA, FANCI, FANCM, NBN, RAD51C, RAD51D, RAD54L 74% of patients with somatic BRCA mutations had an objective response. Response was also noted in patients with mutations in non-BRCA homologous recombination genes (ATM, NBN, RAD51C, and RAD51D). ARIEL3 (Coleman et al., 2017) (20) Phase III High-grade serous ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) (somatic) BRCA BRCA mutated patients had longer PFS compared with the placebo group (13.6 vs 5.4 months, HR = 0.32, 95% CI = 0.24 to 0.42). BRCAnalysis CDx test (Myriad Genetics, Salt Lake City, UT) (germline) (Multiple other genes on NGS) Study Design Cancer type Drug Platform to detect mutations Homologous recombination genes studied Results TOPARP (Mateo et al., 2015) (42) Single-arm phase II Prostate Olaparib GeneRead DNAseq Panel (Qiagen, Germany) BRCA1, BRCA2, ATM, FANCA, CHEK2, PALB2, NBN, HDAC2 16 of 50 patients (33%) had tumor aberrations in DNA-repair genes, with 14 of 16 biomarker-positive patients (88%) having a response to olaparib. ARIEL2 (Swisher et al., 2017) (19) Single-arm phase II Ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) BRCA1, BRCA2, ATM, BRIP1, CHEK2, FANCA, FANCI, FANCM, NBN, RAD51C, RAD51D, RAD54L 74% of patients with somatic BRCA mutations had an objective response. Response was also noted in patients with mutations in non-BRCA homologous recombination genes (ATM, NBN, RAD51C, and RAD51D). ARIEL3 (Coleman et al., 2017) (20) Phase III High-grade serous ovarian Rucaparib Foundation Medicine NGS-based T5a assay (Cambridge, MA) (somatic) BRCA BRCA mutated patients had longer PFS compared with the placebo group (13.6 vs 5.4 months, HR = 0.32, 95% CI = 0.24 to 0.42). BRCAnalysis CDx test (Myriad Genetics, Salt Lake City, UT) (germline) (Multiple other genes on NGS) * CI = confidence interval; HR = hazard ratio; NGS = next-generation sequencing; PARP = poly-ADP ribose polymerase; PFS = progression-free survival. The challenge of interpreting somatic alterations in homologous recombination-related genes is highlighted by phosphatase and tensin homolog (PTEN) and HRD (47). PTEN loss of function mutations confer PARP inhibitor sensitivity in endometrial carcinomas and glioblastomas (48,49), but not in prostate cancer cells (50) and ovarian cancer patients (51). This discrepancy highlights the heterogeneity underlying genome maintenance mechanisms, necessitating context-specific clinical evaluation and validation of specific biomarkers. Furthermore, the low frequency and indeterminate functional relevance of specific mutations in non-BRCA homologous recombination genes add further to the difficulties of a single-gene approach. Consequently, efforts have been made to identify genomic, transcriptomic, proteomic, and functional assays that would be reflective of underlying HRD in tumors. Genomic Scar Assays Cancer genomes often harbor chromosomal aberrations arising from defective DNA repair (52). In BRCA mutant cells, chromosomal spreads reveal increased gross chromosomal rearrangements (23). This leads to the development of assays to evaluate the “genomic scar” left behind by the loss of homologous recombination function, irrespective of which component of the pathway was lost. Current genomic scar assays use a combination of high-throughput genomic profiling techniques including array-based comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) genotyping, and next-generation sequencing (NGS). aCGH of Structural Chromosomal Rearrangements aCGH is a technique that detects genomic copy number variations in tumors (53). Using aCGH genomic profiles, primary breast cancers reveal four distinct subgroups, two of which were enriched for BRCA1/2 deficiency (54). The presence of a BRCA1-like aCGH signature predicted favorable response to platinum vs conventional anthracycline-based therapy (55). Only two-thirds of BRCA1-like tumors harbor either the BRCA1 mutation or promoter methylation, suggesting that the signature captures a wider spectrum of HRD tumors. The BRCA1-like and BRCA2-like profiles were later combined to establish a BRCA-likeCGH score (56) and an assay compatible with formalin-fixed paraffin-embedded (FFPE) samples (57). BRCA-likeCGH was evaluated retrospectively in a breast cancer clinical trial comparing chemotherapy alone with chemotherapy followed by high-dose platinum and autologous stem cell transplant. The study showed no difference in survival for the unselected population, whereas BRCA-likeCGH patients had a statistically significant overall survival (OS) benefit from high-dose platinum-based therapy (hazard ratio [HR] = 0.19, 95% confidence interval [CI] = 0.08 to 0.48). aCGH assays have not been evaluated in the context of PARP inhibition. SNP-Based “Genomic Scar” Assays In 2012, three SNP-based assays were developed to quantify the extent of chromosomal abnormalities—telomeric allelic imbalance (TAI) (58), loss of heterozygosity (LOH) (59), and large-scale transition (LST) (60), jointly termed “genomic scar” assays (Table 3). Each genomic scar assay measures a discrete type of gross genomic aberration, and they were developed using a training cohort of tumors from BRCA1/2 mutated patients. In an in silico analysis of 5371 tumors of 15 cancer types available in the TCGA, cancers where platinum constitutes standard firstline therapy showed increased genomic scar scores (61). Several combination HRD scores have been described (62), with most data for the 3-factor combination scar assay by Myriad Genetics (63). The performance of the combined TAI, LOH, and LST score to detect BRCA mutated cases, with minor adjustments to the definition criteria of the TAI and LST scores, was assessed in a cohort of 215 breast cancer tumors. All three individual scores—LOH, modified TAI, and LST—were statistically significantly associated with BRCA deficiency in all breast cancer samples. In a multivariable model, a combined mean HRD score was statistically significantly associated with BRCA deficiency, at an odds ratio (OR) of 87 (95% CI = 17 to 150). The combination score of LOH, TAI, and LST was also retrospectively validated to predict for response to neoadjuvant platinum therapy in three cohorts of triple-negative breast cancer (TNBC)—cisplatin-1, cisplatin-2, and PrECOG 0105 (64). Recently, the combined HRD score was also shown to predict pathologic complete response (pCR) after anthracycline- and/or taxane-based neoadjuvant chemotherapy in TNBC, irrespective of BRCA mutation status (65), as well as in TNBC patients after paclitaxel, doxorubicin, and bevacizumab with or without carboplatin (66). This combination score, marketed as the myChoice HRD test (Myriad Genetics), was further evaluated in the NOVA study of the PARP inhibitor niraparib in ovarian cancer (17). In the non-gBRCA mutated cohort of NOVA, a preplanned analysis on archival tumor tissue using the myChoice test showed a statistically significant benefit with niraparib treatment for myChoice-positive patients, which was greater than the benefit noted for non-HRD patients. High HRD score (>42) and/or BRCA1/2 mutation has also been shown to be associated with long-term response to olaparib (46). Among the single score assays, only the Foundation medicine LOH assay has been validated prospectively in the phase III setting (ARIEL 3 study) (Table 1). Table 1. Clinical evidence for regulatory approval of PARP inhibitors* Study/first author Design Cancer type Drug Inclusion Results Regulatory approval Kaufman et al. (14) Phase II (nonrandomized) Ovarian, breast, pancreatic, prostate Olaparib Deleterious gBRCA mutation and advanced solid tumor Overall response rate was 26%. Stable disease observed in 42%. FDA approval for maintenance treatment of gBRCA mutated recurrent ovarian cancer. Study 19 (15) Phase II (randomized, double-blind) Ovarian Olaparib Platinum-sensitive relapsed serous ovarian cancer Median PFS for olaparib vs placebo was 11.2 vs 4.3 months, respectively, in BRCA mutated patients and 7.5 vs 5.5 months, respectively, in BRCA wild-type. There was no OS benefit. EMA approval for maintenance in gBRCA mutated ovarian cancer. SOLO2/ENGOT-Ov21 (16) Phase III (randomized, double-blind) Ovarian Olaparib Relapsed ovarian cancer patients with a BRCA mutation, two prior lines of chemotherapy Median PFS for olaparib vs placebo was 19.1 vs 5.5 months, respectively. Additional FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. NOVA (17) Phase III (randomized, double-blind) Ovarian Niraparib Platinum-sensitive ovarian cancer Median PFS for olaparib vs placebo was 21.0 vs 5.5 months, respectively, in gBRCA mutated patients, 12.9 vs 3.8, respectively, in non-gBRCA mutated patients with HRD, and 9.3 vs 3.9 in the non-gBRCA mutated group without HRD. FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. Study 10 (18) Phase I–II (open-label) Ovarian Rucaparib Phase II enrolled platinum-sensitive gBRCA ovarian cancer, two to four prior lines of chemotherapy Objective response rate for gBRCA patients was 59.5%. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL2 (19) Phase II (open-label) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for BRCA mutated and LOH-high subgroups was 12.8 and 5.7 months, respectively, compared with 5.2 months in the LOH-low subgroup. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL3 (20) Phase III (randomized, double-blind) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for rucaparib vs placebo was 16.6 vs 5.4 months, respectively, in BRCA mutated patients and 13.6 vs 5.4, respectively, in the HRD group. FDA approval for advanced ovarian cancer. OlympiAD (21) Phase III (randomized, open label) Breast Olaparib HER2-negative metastatic breast cancer with gBRCA mutation Median PFS for olaparib vs chemotherapy (capecitabine, vinorelbine, eribulin) was 7.0 vs 4.2, respectively. Olaparib showed a higher response rate (59.9% vs 28.8%, olaparib and chemotherapy, respectively). FDA approval for metastatic breast cancer with gBRCA mutations. Study/first author Design Cancer type Drug Inclusion Results Regulatory approval Kaufman et al. (14) Phase II (nonrandomized) Ovarian, breast, pancreatic, prostate Olaparib Deleterious gBRCA mutation and advanced solid tumor Overall response rate was 26%. Stable disease observed in 42%. FDA approval for maintenance treatment of gBRCA mutated recurrent ovarian cancer. Study 19 (15) Phase II (randomized, double-blind) Ovarian Olaparib Platinum-sensitive relapsed serous ovarian cancer Median PFS for olaparib vs placebo was 11.2 vs 4.3 months, respectively, in BRCA mutated patients and 7.5 vs 5.5 months, respectively, in BRCA wild-type. There was no OS benefit. EMA approval for maintenance in gBRCA mutated ovarian cancer. SOLO2/ENGOT-Ov21 (16) Phase III (randomized, double-blind) Ovarian Olaparib Relapsed ovarian cancer patients with a BRCA mutation, two prior lines of chemotherapy Median PFS for olaparib vs placebo was 19.1 vs 5.5 months, respectively. Additional FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. NOVA (17) Phase III (randomized, double-blind) Ovarian Niraparib Platinum-sensitive ovarian cancer Median PFS for olaparib vs placebo was 21.0 vs 5.5 months, respectively, in gBRCA mutated patients, 12.9 vs 3.8, respectively, in non-gBRCA mutated patients with HRD, and 9.3 vs 3.9 in the non-gBRCA mutated group without HRD. FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. Study 10 (18) Phase I–II (open-label) Ovarian Rucaparib Phase II enrolled platinum-sensitive gBRCA ovarian cancer, two to four prior lines of chemotherapy Objective response rate for gBRCA patients was 59.5%. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL2 (19) Phase II (open-label) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for BRCA mutated and LOH-high subgroups was 12.8 and 5.7 months, respectively, compared with 5.2 months in the LOH-low subgroup. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL3 (20) Phase III (randomized, double-blind) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for rucaparib vs placebo was 16.6 vs 5.4 months, respectively, in BRCA mutated patients and 13.6 vs 5.4, respectively, in the HRD group. FDA approval for advanced ovarian cancer. OlympiAD (21) Phase III (randomized, open label) Breast Olaparib HER2-negative metastatic breast cancer with gBRCA mutation Median PFS for olaparib vs chemotherapy (capecitabine, vinorelbine, eribulin) was 7.0 vs 4.2, respectively. Olaparib showed a higher response rate (59.9% vs 28.8%, olaparib and chemotherapy, respectively). FDA approval for metastatic breast cancer with gBRCA mutations. * EMA = European Medicines Agency; FDA = Food and Drug Administration; gBRCA = germline BRCA1/2; HER2 = human epidermal growth factor receptor 2; HR = hazard ratio; HRD = homologous recombination deficiency; LOH = loss of heterozygosity; OS = overall survival; PARP = poly-ADP ribose polymerase; PFS = progression-free survival. Table 1. Clinical evidence for regulatory approval of PARP inhibitors* Study/first author Design Cancer type Drug Inclusion Results Regulatory approval Kaufman et al. (14) Phase II (nonrandomized) Ovarian, breast, pancreatic, prostate Olaparib Deleterious gBRCA mutation and advanced solid tumor Overall response rate was 26%. Stable disease observed in 42%. FDA approval for maintenance treatment of gBRCA mutated recurrent ovarian cancer. Study 19 (15) Phase II (randomized, double-blind) Ovarian Olaparib Platinum-sensitive relapsed serous ovarian cancer Median PFS for olaparib vs placebo was 11.2 vs 4.3 months, respectively, in BRCA mutated patients and 7.5 vs 5.5 months, respectively, in BRCA wild-type. There was no OS benefit. EMA approval for maintenance in gBRCA mutated ovarian cancer. SOLO2/ENGOT-Ov21 (16) Phase III (randomized, double-blind) Ovarian Olaparib Relapsed ovarian cancer patients with a BRCA mutation, two prior lines of chemotherapy Median PFS for olaparib vs placebo was 19.1 vs 5.5 months, respectively. Additional FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. NOVA (17) Phase III (randomized, double-blind) Ovarian Niraparib Platinum-sensitive ovarian cancer Median PFS for olaparib vs placebo was 21.0 vs 5.5 months, respectively, in gBRCA mutated patients, 12.9 vs 3.8, respectively, in non-gBRCA mutated patients with HRD, and 9.3 vs 3.9 in the non-gBRCA mutated group without HRD. FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. Study 10 (18) Phase I–II (open-label) Ovarian Rucaparib Phase II enrolled platinum-sensitive gBRCA ovarian cancer, two to four prior lines of chemotherapy Objective response rate for gBRCA patients was 59.5%. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL2 (19) Phase II (open-label) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for BRCA mutated and LOH-high subgroups was 12.8 and 5.7 months, respectively, compared with 5.2 months in the LOH-low subgroup. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL3 (20) Phase III (randomized, double-blind) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for rucaparib vs placebo was 16.6 vs 5.4 months, respectively, in BRCA mutated patients and 13.6 vs 5.4, respectively, in the HRD group. FDA approval for advanced ovarian cancer. OlympiAD (21) Phase III (randomized, open label) Breast Olaparib HER2-negative metastatic breast cancer with gBRCA mutation Median PFS for olaparib vs chemotherapy (capecitabine, vinorelbine, eribulin) was 7.0 vs 4.2, respectively. Olaparib showed a higher response rate (59.9% vs 28.8%, olaparib and chemotherapy, respectively). FDA approval for metastatic breast cancer with gBRCA mutations. Study/first author Design Cancer type Drug Inclusion Results Regulatory approval Kaufman et al. (14) Phase II (nonrandomized) Ovarian, breast, pancreatic, prostate Olaparib Deleterious gBRCA mutation and advanced solid tumor Overall response rate was 26%. Stable disease observed in 42%. FDA approval for maintenance treatment of gBRCA mutated recurrent ovarian cancer. Study 19 (15) Phase II (randomized, double-blind) Ovarian Olaparib Platinum-sensitive relapsed serous ovarian cancer Median PFS for olaparib vs placebo was 11.2 vs 4.3 months, respectively, in BRCA mutated patients and 7.5 vs 5.5 months, respectively, in BRCA wild-type. There was no OS benefit. EMA approval for maintenance in gBRCA mutated ovarian cancer. SOLO2/ENGOT-Ov21 (16) Phase III (randomized, double-blind) Ovarian Olaparib Relapsed ovarian cancer patients with a BRCA mutation, two prior lines of chemotherapy Median PFS for olaparib vs placebo was 19.1 vs 5.5 months, respectively. Additional FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. NOVA (17) Phase III (randomized, double-blind) Ovarian Niraparib Platinum-sensitive ovarian cancer Median PFS for olaparib vs placebo was 21.0 vs 5.5 months, respectively, in gBRCA mutated patients, 12.9 vs 3.8, respectively, in non-gBRCA mutated patients with HRD, and 9.3 vs 3.9 in the non-gBRCA mutated group without HRD. FDA approval for maintenance treatment of recurrent ovarian cancer, regardless of gBRCA mutation status. Study 10 (18) Phase I–II (open-label) Ovarian Rucaparib Phase II enrolled platinum-sensitive gBRCA ovarian cancer, two to four prior lines of chemotherapy Objective response rate for gBRCA patients was 59.5%. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL2 (19) Phase II (open-label) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for BRCA mutated and LOH-high subgroups was 12.8 and 5.7 months, respectively, compared with 5.2 months in the LOH-low subgroup. FDA acceptance of priority review for treatment of advanced gBRCA mutated ovarian cancer. ARIEL3 (20) Phase III (randomized, double-blind) Ovarian Rucaparib Platinum-sensitive ovarian cancer, two prior lines of chemotherapy Median PFS for rucaparib vs placebo was 16.6 vs 5.4 months, respectively, in BRCA mutated patients and 13.6 vs 5.4, respectively, in the HRD group. FDA approval for advanced ovarian cancer. OlympiAD (21) Phase III (randomized, open label) Breast Olaparib HER2-negative metastatic breast cancer with gBRCA mutation Median PFS for olaparib vs chemotherapy (capecitabine, vinorelbine, eribulin) was 7.0 vs 4.2, respectively. Olaparib showed a higher response rate (59.9% vs 28.8%, olaparib and chemotherapy, respectively). FDA approval for metastatic breast cancer with gBRCA mutations. * EMA = European Medicines Agency; FDA = Food and Drug Administration; gBRCA = germline BRCA1/2; HER2 = human epidermal growth factor receptor 2; HR = hazard ratio; HRD = homologous recombination deficiency; LOH = loss of heterozygosity; OS = overall survival; PARP = poly-ADP ribose polymerase; PFS = progression-free survival. Table 3. SNP-based “genomic scar” assays* Assay Description Biological rationale Retrospective validation Prospective validation Telomeric allelic imbalance Subchromosomal regions displaying allelic imbalance extending to the telomere but not crossing the centromere (58) HRD leads to aberrant chromosomal end fusions due to inappropriate end joining, which would then lead to an allelic-imbalance after breakage during mitosis. Neoadjuvant cisplatin for TNBC showed a statistically significant correlation between higher levels of TAI and response. TCGA data show elevated TAI in gBRCA mutated ovarian cancer (58). Not as single assay (combination studies only) Loss of heterozygosity Analysis of the number of regions with LOH, of more than 15 Mb and shorter than the whole chromosome (59) HRD results in uniparental disomy or LOH due to inaccurate repair of sister chromatids during the S/G2 phase of the cell cycle. Higher levels of LOH are seen in BRCA-deficient ovarian cancers than in sporadic cancers (59,67) and are associated with lower chemotherapy resistance rates and longer PFS (68). In the ARIEL2 study, there was a longer PFS in the LOH-high subgroup compared with LOH-low (HR= 0.62, 95% CI = 0.42 to 0.90) (19). Large-scale transition Chromosomal breaks of more than 10 Mb (after exclusion of regions shorter than 3 Mb) (60) Large structural chromosomal aberrations arise in HRD cells due to inappropriate recombination between chromosomal segments. BRCA/RAD51C-inactivated breast cancers show higher LST than sporadic cancers (60,69). Not as single assay (combination studies only) Assay Description Biological rationale Retrospective validation Prospective validation Telomeric allelic imbalance Subchromosomal regions displaying allelic imbalance extending to the telomere but not crossing the centromere (58) HRD leads to aberrant chromosomal end fusions due to inappropriate end joining, which would then lead to an allelic-imbalance after breakage during mitosis. Neoadjuvant cisplatin for TNBC showed a statistically significant correlation between higher levels of TAI and response. TCGA data show elevated TAI in gBRCA mutated ovarian cancer (58). Not as single assay (combination studies only) Loss of heterozygosity Analysis of the number of regions with LOH, of more than 15 Mb and shorter than the whole chromosome (59) HRD results in uniparental disomy or LOH due to inaccurate repair of sister chromatids during the S/G2 phase of the cell cycle. Higher levels of LOH are seen in BRCA-deficient ovarian cancers than in sporadic cancers (59,67) and are associated with lower chemotherapy resistance rates and longer PFS (68). In the ARIEL2 study, there was a longer PFS in the LOH-high subgroup compared with LOH-low (HR= 0.62, 95% CI = 0.42 to 0.90) (19). Large-scale transition Chromosomal breaks of more than 10 Mb (after exclusion of regions shorter than 3 Mb) (60) Large structural chromosomal aberrations arise in HRD cells due to inappropriate recombination between chromosomal segments. BRCA/RAD51C-inactivated breast cancers show higher LST than sporadic cancers (60,69). Not as single assay (combination studies only) * CI = confidence interval; gBRCA = germline BRCA1/2; HR = hazard ratio; HRD = homologous recombination deficiency; LOH = loss of heterozygosity; LST = large-scale transition; PFS = progression-free survival; SNP = single nucleotide polymorphism; TAI = telomeric allelic imbalance; TCGA = The Cancer Genome Atlas; TNBC = triple-negative breast cancer Table 3. SNP-based “genomic scar” assays* Assay Description Biological rationale Retrospective validation Prospective validation Telomeric allelic imbalance Subchromosomal regions displaying allelic imbalance extending to the telomere but not crossing the centromere (58) HRD leads to aberrant chromosomal end fusions due to inappropriate end joining, which would then lead to an allelic-imbalance after breakage during mitosis. Neoadjuvant cisplatin for TNBC showed a statistically significant correlation between higher levels of TAI and response. TCGA data show elevated TAI in gBRCA mutated ovarian cancer (58). Not as single assay (combination studies only) Loss of heterozygosity Analysis of the number of regions with LOH, of more than 15 Mb and shorter than the whole chromosome (59) HRD results in uniparental disomy or LOH due to inaccurate repair of sister chromatids during the S/G2 phase of the cell cycle. Higher levels of LOH are seen in BRCA-deficient ovarian cancers than in sporadic cancers (59,67) and are associated with lower chemotherapy resistance rates and longer PFS (68). In the ARIEL2 study, there was a longer PFS in the LOH-high subgroup compared with LOH-low (HR= 0.62, 95% CI = 0.42 to 0.90) (19). Large-scale transition Chromosomal breaks of more than 10 Mb (after exclusion of regions shorter than 3 Mb) (60) Large structural chromosomal aberrations arise in HRD cells due to inappropriate recombination between chromosomal segments. BRCA/RAD51C-inactivated breast cancers show higher LST than sporadic cancers (60,69). Not as single assay (combination studies only) Assay Description Biological rationale Retrospective validation Prospective validation Telomeric allelic imbalance Subchromosomal regions displaying allelic imbalance extending to the telomere but not crossing the centromere (58) HRD leads to aberrant chromosomal end fusions due to inappropriate end joining, which would then lead to an allelic-imbalance after breakage during mitosis. Neoadjuvant cisplatin for TNBC showed a statistically significant correlation between higher levels of TAI and response. TCGA data show elevated TAI in gBRCA mutated ovarian cancer (58). Not as single assay (combination studies only) Loss of heterozygosity Analysis of the number of regions with LOH, of more than 15 Mb and shorter than the whole chromosome (59) HRD results in uniparental disomy or LOH due to inaccurate repair of sister chromatids during the S/G2 phase of the cell cycle. Higher levels of LOH are seen in BRCA-deficient ovarian cancers than in sporadic cancers (59,67) and are associated with lower chemotherapy resistance rates and longer PFS (68). In the ARIEL2 study, there was a longer PFS in the LOH-high subgroup compared with LOH-low (HR= 0.62, 95% CI = 0.42 to 0.90) (19). Large-scale transition Chromosomal breaks of more than 10 Mb (after exclusion of regions shorter than 3 Mb) (60) Large structural chromosomal aberrations arise in HRD cells due to inappropriate recombination between chromosomal segments. BRCA/RAD51C-inactivated breast cancers show higher LST than sporadic cancers (60,69). Not as single assay (combination studies only) * CI = confidence interval; gBRCA = germline BRCA1/2; HR = hazard ratio; HRD = homologous recombination deficiency; LOH = loss of heterozygosity; LST = large-scale transition; PFS = progression-free survival; SNP = single nucleotide polymorphism; TAI = telomeric allelic imbalance; TCGA = The Cancer Genome Atlas; TNBC = triple-negative breast cancer Mutational Signatures Cancer types bear distinct mutational signatures, that is, mutational patterns (as opposed to isolated detection of mutations at selected loci), likely acquired through distinct patterns of mutagen exposure and genomic instability, which can be identified using NGS. The overall genome-wide mutation burden is associated with improved survival after chemotherapy in BRCA mutated ovarian cancers (70). Interestingly, BRCA2 mutated ovarian cancers are associated with a higher mutator phenotype than BRCA1 mutant cancers (71). This underscores a need for multidimensional scores to unravel the complexity of HRD patterns. The mutational burden assay was refined by combining somatic mutation count with copy number variations to improve the identification of BRCA mutated ovarian cancer patients from the TCGA (72). The incorporation of pattern of mutations, in addition to mutational burden, has led to the development of specific “signatures” of mutations in cancer, based on base substitution patterns. One of these, “signature 3,” described by Alexandrov et al., is attributed to underlying HRD (73) and has been shown to exist in several cancers, including breast, ovarian, prostate, and gastric. HRDetect, a weighted model of mutational signatures, has recently demonstrated good performance in predicting BRCA deficiency (defined as somatic or germline mutations and BRCA1 promoter hypermethylation) in breast, ovarian, and pancreatic cancers (74). This model incorporates a weighted score of microhomology-mediated deletions, base substitution/rearrangement signatures, and the HRD index based on genomic scars. HRDetect identifies BRCA-deficient tumors at high sensitivity and specificity from FFPE samples (area under the receiver operating characteristic curve = 0.98). Additionally, one-third of the HRD-high score cases were BRCA wild-type and did not associate with known mutations in other homologous recombination genes. However, the HRDetect algorithm has yet to be validated in a clinical trial of PARP inhibition. Limitations of Genomic Scar Assays Limitations of the genomic scar assays were highlighted by the inability of the Myriad myChoice assay to predict for platinum sensitivity in the TNT study of docetaxel or carboplatin in metastatic TNBC patients (32). Prior anthracycline exposure in this setting may contribute to the inaccuracy of the assay, as the emergence of resistance pathways would not remove the existing “scar” of prior HRD. This is substantiated by the improved performance of this assay in the neoadjuvant setting (64). The predictive potential of genomic scar HRD assays could therefore be undermined by mechanisms dynamically affecting the homologous recombination pathway and drug accumulation. BRCA1/2 (30,75), and recently RAD51C/D (76) and PALB2 (77), reversions or secondary mutations have been described to restore homologous recombination. A BRCA mutation may have initially imprinted a genomic HRD scar signature, but, upon reversion, the tumor would regain homologous recombination functionality even if the HRD scar was still detectable. This is particularly relevant in ovarian cancer, where approximately half of all platinum-resistant BRCA mutated tumors eventually develop restoration of BRCA function in response to platinum therapy (78,79). Restoration of BRCA2 function was also reported in pancreatic cancer in response to the PARP inhibitor olaparib (80). Other BRCA-dependent resistance mechanisms include stabilization of BRCT domain mutants of BRCA1 by HSP90 (81), alternative splicing (82), or alternative translation initiation of BRCA1 (83). BRCA1-independent mechanisms governing PARP inhibitor resistance leverage on promoting genomic stability at stalled replication forks (84,85) or modulation of double-strand DNA break end resection (86,87). Finally, acquired or intrinsic resistance is frequently mediated by membrane transporters (88), as described in olaparib resistance in mouse models due to upregulation of the efflux pump P-glycoprotein (89). These mechanisms are not captured in genomic scar HRD assays. A negative myChoice HRD scar assay does not also exclude HRD. In the phase III NOVA clinical trial of maintenance therapy of PARP inhibitor niraparib in platinum-sensitive recurrent ovarian cancer (17), niraparib improved progression-free survival (PFS), even in the cohort classified as HRD proficient (6.9 vs 3.8 months; HR = 0.58, 95% CI = 0.36 to 0.92). NOVA highlights challenges with the use of SNP-based genomic scar assays to exclude patients for PARP inhibitor therapy, especially in a platinum-sensitive setting. For the caveats listed above, assays that report a “real-time” index of HR in the tumor sample would be of clinical interest and value. Real-time Indicators of HRD Unlike genomic alterations, which are reflective of past events, RNA and proteins vary dynamically in quantity and localization during cellular processes. They are therefore postulated to provide a current snapshot of the state of HRD in a given tumor sample. Transcriptional Profiles Gene expression profiling (GEP) captures a current transcriptional state of a tumor, with platforms such as OncotypeDx already in clinical use for the selection of patients for adjuvant chemotherapy (90). GEP assays, including the 93-gene Chemotherapy Response Profile (CRP), have similarly been retrospectively evaluated, with promising results in the context of platinum responses in ovarian cancer (91–93). These GEP panels, though not exclusively focused on homologous recombination, have interrogated genes that regulate apoptosis, cell cycle entry, and DNA repair. BRCA germline mutated ovarian tumors show a distinct GEP from sporadic tumors and normal ovarian surface epithelium (94). BRCA profiles are present in some sporadic tumors (95), however, highlighting the potential value of such GEP assays as a broader net for HRD in cancer. A 60-gene signature clusters tumors into BRCA-like and non-BRCA-like (96), and this BRCAness profile could predict platinum sensitivity even within a BRCA mutant population. Importantly, the development of platinum resistance during the course of therapy was reflected in the change of BRCAness profile in two out of four patients, highlighting the dynamic nature of these assays. In breast cancer, an FFPE-compatible 44-gene microarray-based assay, termed the DDR deficiency assay (97), predicted complete pathologic response after neoadjuvant chemotherapy (OR = 4.0) and improved five-year relapse-free survival (HR = 0.37, 95% CI = 0.15 to 0.88). A 77-gene expression profile termed BRCA1ness was developed based on a cohort of TNBC associated with a known BRCA1-like aCGH profile (55,98). Genes included in this profile constitute signatures of DNA replication, recombination and repair, cellular function/maintenance, cellular assembly, and cell cycle, and also metabolic signatures of serine, glycine, and histamine biosynthesis. The BRCA1ness GEP was evaluated in the phase II I-SPY 2 study, where human epidermal growth factor receptor 2–negative breast cancer patients were randomly assigned to receive paclitaxel with or without concurrent veliparib and carboplatin (V-C). Patients with a BRCA1ness GEP showed a statistically significantly higher proportion of pCR at an odds ratio of 3.2 only in the V-C treated group. At present, none of the above GEP assays have been specifically assessed in the context of PARP inhibition. Their comparative performance in retrospective clinical trial cohorts and compatibility with FFPE samples will be critical in further development. Protein Expression The expression of tumor suppressor proteins involved in homologous recombination has been analyzed in cancer tissue using immunohistochemical approaches, typically in retrospective studies. Immunohistochemical analyses of the BRCA1 and 2 proteins have been hindered by the absence of validated reagents and low expression of these proteins. Among non-BRCA DDR proteins, ataxia-telangiectasia mutated kinase (ATM) has been evaluated most extensively in clinical studies of PARP inhibition. ATM operates upstream of homologous recombination in the DNA damage response, and depletion of this protein is associated with PARP inhibitor sensitivity in vitro (38,99–101). ATM loss as measured by immunohistochemistry (IHC) has been described in various tumor groups, including gastric (102), colon (103), and breast (104) cancer. In gastric cancer, ATM loss occurs in about 20% of the population (99,102). A randomized phase II study of paclitaxel+olaparib compared with paclitaxel+placebo in gastric cancer demonstrated OS benefit in the ATM loss population (105). Unfortunately, this was not borne out in the follow-up randomized phase III GOLD study (106). The loss of ATM can be partially compensated for by other DNA repair pathways, which were not measured in the GOLD trial samples. Single protein IHC assays are likely to be superseded by novel approaches, such as multispectral microscopy, tissue mass cytometry, and digital spatial profiling, which provide multiplexed information in the histological analysis of proteins. The application of multiplex analyses of DNA repair proteins in clinical trial material may herald new insights into biomarkers of HRD. Functional Assays Phenotypic or functional assays are appealing, given the challenges of measuring all proteins of interest within a pathway for each cancer sample. For example, a functional assay of HRD should ideally measure a single downstream event that would reflect proficiency of multiple upstream components of homologous recombination. Quantification of RAD51 foci is a prototypical example of a functional HRD assay. In the S/G2 phases of the cell cycle, when the sister chromatid is available for recombination to occur, RAD51 forms distinct subnuclear foci after DNA-damaging insults. The inability of cells to form RAD51 foci is a common feature of HRD. It potentially provides a global read-out of HRD without defining the underlying cause of homologous recombination deficiency or reversion, as many of the factors influencing these might still be unknown. RAD51 foci in S/G2 cells were evaluated in FFPE breast cancer tissue 24 hours postchemotherapy (107) using a counterstain to geminin, a marker for S and G2 cells (108). RAD51-low score was more common in TNBC than in other subtypes, with higher pCR rates (33% vs 3%). A major challenge to be overcome is the reliable quantitation of “foci” in FFPE samples, where fixation artefacts are common. Furthermore, the baseline level of RAD51 (and other DDR) foci is not as relevant as the relative increase in their numbers upon DNA damage. Therefore, another challenge with such assays is the lack of availability of post-treatment biopsies in routine clinical practice. Ex vivo experiments offer a workaround, for example, in the investigation of ionizing radiation–induced RAD51 foci in organotypic breast cancer samples (109) or primary cultures of ascites in epithelial ovarian cancer patients with rucaparib treatment ex vivo (110). While these studies point to the potential of RAD51 as a composite marker of homologous recombination, several technical and practical challenges curtail its clinical utility in its current form. The tissue context, modality of DNA damage used, timing of RAD51 foci assessment (111), and resolution of the microscopy system all account for the large differences of foci assessment between these studies. False-negative RAD51 foci formation assays may also occur with defects operating downstream of RAD51-ssDNA filament formation, such as depletion of RAD51-associated protein 1 (RAD51AP1) (112) and polymerase eta disruption (113). Cells with loss of the upstream DDR components of the MRN complex (114) and ATM form RAD51 foci in vitro, but display defects in homologous recombination (115,116). Finally, resistance to PARP inhibition in mouse models is associated with a persistent RAD51 focus formation defect, which points to a critical role for replication fork stabilization in PARP inhibitor sensitivity (84). Therefore, at present, RAD51 foci formation remains a biologically useful marker that is not easily quantifiable in clinical material. A clinically measurable composite marker of replication fork stabilization may, however, offer a promising phenotypic marker of PARP inhibitor sensitivity. Conclusions HRD is a bona fide anticancer target where multiple therapeutic modalities have demonstrated varying degrees of efficacy, including chemotherapy and DNA repair enzyme inhibition. The aforementioned biomarkers have provided tantalizing hints of their relevance in specific tumor types. Nonetheless, the development of a generic touchstone biomarker for HRD will be key to expanding the therapeutic utility of HRD-targeting agents across a broad spectrum of tumor types. Given the complexity of the homologous recombination pathway, it is unlikely that one single biomarker will suffice. From a practical standpoint, clinical surrogate biomarkers like platinum sensitivity are likely to prevail for the foreseeable future, although these are neither ideal nor sufficiently comprehensive. Primary platinum resistance, for example, may be mediated by mechanisms that are independent of homologous recombination status, and the inclusion of only platinum-sensitive patients will deny a subgroup of homologous recombination-deficient patients the possible benefits of PARP inhibition (Figure 1A). In all likelihood, composite HRD scores involving two or more biomarker strategies will be eventually be required to define “HRDness.” An example for such a score could be “platinum sensitivity OR HRDetect positivity AND decreased replication fork stability.” Such techniques will only be suitable for clinical application if they can provide rapid and precise readouts. The nature of the genomic instability in HRD tumor cells also confers accelerated capacity for therapeutic evasion via clonal evolution. As such, a cornerstone of success in targeting HRD will be to ensure that the optimal therapeutic strategy is applied as early as possible in the course of a patient’s treatment. The difficulties of validating any biomarker approach will be compounded by tumor heterogeneity and clonal evolution in late stages of the disease and treatment. Consequently, this implies a need for constant re-evaluation of tumor homologous recombination status in patients for whom HRD-targeted drugs are being considered. In this context, HRD biomarkers based on tissue biopsy techniques that enable serial and multiple spatially distinct samples or liquid biopsies may be the key to surmounting these hurdles. Ultimately, overcoming challenges of identifying the right patients at the right time for the right therapeutic strategy will allow the full potential of HRD-directed precision medicine to be realized. Funding ADJ and DSPT are recipients of the National Medical Research Council Transition Award and the Clinician-Scientist Award, respectively. RS is supported by the MOH Healthcare Research Scholarship. MMH is a recipient of the Cancer Science Institute of Singapore PhD Graduate Scholarship. This work is supported by the Singapore Ministry of Health's National Medical Research Council under a clinician-scientist grant to DSPT (NMRC/CIRG/1400/2014). Notes Affiliations of authors: Cancer Science Institute of Singapore (MMH, DSPT, ADJ) and Department of Haematology-Oncology (RS, DSPT, ADJ), National University Hospital, Singapore. The funders had no role in the writing of this Commentary or the decision to submit it for publication. The authors wish to thank Celestina Chin for proofreading the manuscript, Piotr Garstecki for help with editing of figures, and Prof. Stanley Kaye for his helpful discussions and insight. Conflicts of interest: ADJ: honoraria from MSD; DSPT: honoraria from AstraZeneca, Roche, and MSD and research funding from AstraZeneca, Bayer, and Karyopharm. References 1 Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer . 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Journal

JNCI: Journal of the National Cancer InstituteOxford University Press

Published: May 18, 2018

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